2′-fluoronucleosides

ABSTRACT

A class of 2′-fluoro-nucleoside compounds are disclosed which are useful in the treatment of hepatitis B infection, hepatitis C infection, HIV and abnormal cellular proliferation, including tumors and cancer. The compounds have the general formula: 
                         
wherein
         Base is a purine or pyrimidine base;   R 1  is OH, H, OR 3 , N 3 , CN, halogen, including F, or CF 3 , lower alkyl, amino, lower alkylamino, di(lower)alkylamino, or alkoxy, and base refers to a purine or pyrimidine base;   R 2  is H, phosphate, including monophosphate, diphosphate, triphosphate, or a stabilized phosphate prodrug; acyl, or other pharmaceutically acceptable leaving group which when administered in vivo, is capable of providing a compound wherein R 2  is H or phosphate; sulfonate ester including alkyl or aryalkyl sulfonyl including methanesulfonyl, benzyl, wherein the phenyl group is optionally substituted with one or more substituents as described in the definition of aryl given above, a lipid, an amino acid, peptide, or cholesterol; and   R 3  is acyl, alkyl, phosphate, or other pharmaceutically acceptable leaving group which when administered in vivo, is capable of being cleaved to the parent compound, or a pharmaceutically acceptable salt thereof.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of application U.S. Ser. No.10/796,529 filed Mar. 8, 2004 now U.S. Pat No. 7,307,065, which is acontinuation of U.S. Ser. No. 10/061,128, filed Jan. 30, 2002, now U.S.Pat. No. 6,911,424, which is a continuation of application of U.S. Ser.No. 09/257,130, filed Feb. 25, 1999, now U.S. Pat. No. 6,348,587, whichclaims benefit of provisional application U.S. Ser. No. 60/075,893,filed on Feb. 25, 1998, and provisional application U.S. Ser. No.60/080,569, filed on Apr. 3, 1998.

The invention described herein was made with Government support undergrant Nos. A132351 and A128931 awarded by the National Institute ofHealth. The United States Government has certain rights to thisinvention.

BACKGROUND OF THE INVENTION

Synthetic nucleosides such as 5-iodo-2′-deoxyuridine and5-fluoro-2′-deoxyuridine have been used for the treatment of cancer andherpes viruses for a number of years. Since the 1980's, syntheticnucleosides have also been a focus of interest for the treatment of HIV,hepatitis, and Epstein-Barr viruses.

In 1981, acquired immune deficiency syndrome (AIDS) was identified as adisease that severely compromises the human immune system, and thatalmost without exception leads to death. In 1983, the etiological causeof AIDS was determined to be the human immunodeficiency virus (HIV). In1985, it was reported that the synthetic nucleoside3′-azido-3′-deoxythymidine (AZT) inhibits the replication of humanimmunodeficiency virus. Since then, a number of other syntheticnucleosides, including 2′,3′-dideoxyinosine (DDI), 2′,3′-dideoxycytidine(DDC), and 2′,3′-dideoxy-2′,3′-didehydrothymidine (D4T), have beenproven to be effective against HIV. After cellular phosphorylation tothe 5′-triphosphate by cellular kinases, these synthetic nucleosides areincorporated into a growing strand of viral DNA, causing chaintermination due to the absence of the 3′-hydroxyl group. They can alsoinhibit the viral enzyme reverse transcriptase.

The success of various synthetic nucleosides in inhibiting thereplication, of HIV in vivo or in vitro has led a number of researchersto design and test nucleosides that substitute a heteroatom for thecarbon atom at the 3′-position of tire nucleoside. European PatentApplication Publication No. 0 337 713 and U.S. Pat. No. 5,041,449,assigned to BioChem Pharma, Inc., disclose racemic2-substituted-4-substituted-1,3-dioxolanes that exhibit antiviralactivity. U.S. Pat. No. 5,047,407 and European Patent Application No. 0382 526, also assigned to BioChem Pharma, Inc., disclose that a numberof racemic 2-substituted-5-substituted-1,3-oxathiolane nucleosides haveantiviral activity, and specifically report that the racemic mixture of2-hydroxymethyl-5-(cytosin-1-yl)-1,3-oxathiolane (referred to below asBCH-189) has approximately the same activity against HIV as AZT, withlittle toxicity. The (−)-enantiomer of the racemate BCH-189, known as3TC, which is covered by U.S. Pat. No. 5,539,116 to Liotta et al., iscurrently sold for the treatment of HIV in combination with AZT inhumans in the U.S.

It has also been disclosed thatcis-2-hydroxymethyl-5-(5-fluorocytosin-1-yl)-1,3-oxathiolane (“FTC”) haspotent HIV activity. Schinazi, etal. , “Selective Inhibition of Humanimmunodeficiency viruses by Racemates and Enantiomers ofcis-5-Fluoro-1-[2-(Hydroxymethyl)-1,3-Oxathiolane-5-yl]Cytosine”Antimicrobial Agents and Chemotherapy, November 1992, pp. 2423-2431. Seealso U.S. Pat. No. 5,210,085; WO 91/11186, and WO 92/14743.

Another virus that causes a serious human health problem is thehepatitis B virus (referred to below as “HBV”). HBV is second only totobacco as a cause of human cancer. The mechanism by which HBV inducescancer is unknown. It is postulated that it may directly trigger tumordevelopment, or indirectly trigger tumor development through chronicinflammation, cirrhosis, and cell regeneration associated with theinfection.

After a two to six month incubation period in which the host is unawareof the infection, HBV infection can lead to acute hepatitis and liverdamage, that causes abdominal pain, jaundice, and elevated blood levelsof certain enzymes. HBV can cause fulminant hepatitis, a rapidlyprogressive, often fatal form of the disease in which massive sectionsof the liver are destroyed.

Patients typically recover from acute hepatitis. In some patients,however, high levels of viral antigen persist in the blood for anextended, or indefinite, period, causing a chronic infection. Chronicinfections can lead to chronic persistent hepatitis. Patients infectedwith chronic persistent HBV are most common in developing countries. Bymid-1991, there were approximately 225 million chronic carriers of HBVin Asia alone, and worldwide, almost 300 million carriers. Chronicpersistent hepatitis can cause fatigue, cirrhosis of the liver, andhepatocellular carcinoma, a primary liver cancer.

In western industrialized countries, high risk groups for HBV infectioninclude those in contact with HBV carriers or their blood samples. Theepidemiology of HBV is very similar to that of acquired immunedeficiency syndrome, which accounts for why HBV infection is commonamong patients infected with HIV or AIDS. However, HBV is morecontagious than HIV.

Both FTC and 3TC exhibit activity against HBV. Furman, et al, “TheAnti-Hepatitis B Virus Activities, Cytotoxicities, and Anabolic Profilesof the (−) and (+) Enantiomers ofcis-5-Fluoro-1-[2-(Hydroxymethyl)-1,3-oxathiolane-5-yl]-Cytosine”Antimicrobial Agents and Chemotherapy, December 1992, pp. 2686-2692; andCheng, et al., Journal of Biological Chemistry, Volume 267(20), pp.13938-13942 (1992).

A human serum-derived vaccine has been developed to immunize patientsagainst HBV. While it has been found effective, production of thevaccine is troublesome because the supply of human serum from chroniccarriers is limited, and the purification procedure is long andexpensive. Further, each batch of vaccine prepared from different serummust be tested in chimpanzees to ensure safety. Vaccines have also beenproduced through genetic engineering. Daily treatments witha-interferon, a genetically engineered protein, has also shown promise.

Hepatitis C virus (“HCV”) is the major causative agent forpost-transfusion and for sporadic non A, non B hepatitis (Alter. H. J.(1990) J. Gastro. Hepatol 1:78-94; Dienstag, J. L. (1983) Gastro85:439-462). Despite improved screening, HCV still accounts for at least25% of the acute viral hepatitis in many countries (Alter, H. J. (1990)supra; Dienstag, J. L. (1983) supra; Alter M. J. et al. (1990a) J.A.M.A.264:2231-2235; Alter M. J. et al (1992) N. Engl. J. Med. 327:1899-1905;Alter, M. J. et al. (1990b) N. Engl. J. Med. 321:1494-1500). Infectionby HCV is Insidious in a high proportion of chronically Infected (andinfectious) carriers who may not experience clinical symptoms for manyyears. The high rate of progression of acute infection to chronicInfection (70-100%) and liver disease (>50%), its world-widedistribution and lack of a vaccine make HCV a significant cause ofmorbidity and mortality.

A tumor is an unregulated, disorganized proliferation of cell growth. Atumor is malignant, or cancerous, if it has the properties ofinvasiveness and metastasis. Invasiveness refers to the tendency of atumor to enter surrounding tissue, breaking through the basal laminasthat define the boundaries of the tissues, thereby often entering thebody's circulatory system. Metastasis refers to the tendency of a tumorto migrate to other areas of the body and establish areas ofproliferation away from the site of initial appearance.

Cancer is now the second leading cause of death in the United States.Over 8,000,000 persons in the United States have been diagnosed withcancer, with 1,208,000 new diagnoses expected in 1994. Over 500,000people die annually from the disease in this country.

Cancer is not fully understood on the molecular level. It is known thatexposure of a cell to a carcinogen such as certain viruses, certainchemicals, or radiation, leads to DNA alteration that inactivates a“suppressive” gene or activates an “oncogene”. Suppressive genes aregrowth regulatory genes, which upon mutation, can no longer control cellgrowth. Oncogenes are initially normal genes (called prooncongenes) thatby mutation or altered context of expression become transforming genes.The products of transforming genes cause inappropriate cell growth. Morethan twenty different normal cellular genes can become oncogenes bygenetic alteration. Transformed cells differ from normal cells in manyways, including cell morphology, cell-to-cell interactions, membranecontent, cytoskeletal structure, protein secretion, gene expression andmortality (transformed cells can grow indefinitely).

All of the various cell types of the body can be transformed into benignor malignant tumor cells. The most frequent tumor site is lung, followedby colorectal, breast, prostate, bladder, pancreas, and then ovary.Other prevalent types of cancer include leukemia, central nervous systemcancers, including brain cancer, melanoma, lymphoma, erythroleukemia,uterine cancer, and head and neck cancer.

Cancer is now primarily treated with one or a combination of three yearsof therapies: surgery, radiation, and chemotherapy. Surgery involves thebulk removal of diseased tissue. While surgery is sometimes effective inremoving tumors located at certain sites, for example, in the breast,colon, and skin, it cannot be used in the treatment of tumors located inother areas, such as the backbone, nor in the treatment of disseminatedneoplastic conditions such as leukemia.

Chemotherapy involves the disruption of cell replication or cellmetabolism. It is used most often in the treatment of leukemia, as wellas breast, lung, and testicular cancer.

There are five major classes of chemotherapeutic agents currently in usefor the treatment of cancer: natural products and their derivatives;anthracyclines: alkylating agents; antiproliferatives (also calledantimetabolites); and hormonal agents. Chemotherapeutic agents are oftenreferred to as antineoplastic agents.

The alkylating agents are believed to act by alkylating andcross-linking guanine and possibly other bases in DNA, arresting celldivision. Typical alkylating agents include nitrogen mustards,ethyleneimine compounds, alkyl sulfates, cisplatin, and variousnitrosoureas. A disadvantage with these compounds is that they not onlyattach malignant cells, but also other cells which are naturallydividing, such as those of bone marrow, skin, gastrointestinal mucosa,and fetal tissue.

Antimetabolites are typically reversible or irreversible enzymeinhibitors, or compounds that otherwise interfere with the replication,translation or transcription of nucleic acids.

Several synthetic nucleosides have been identified that exhibitanticancer activity. A well known nucleoside derivative with stronganticancer activity is 5-fluorouracil. 5-Fluorouracil has been usedclinically in the treatment of malignant tumors, including, for example,carcinomas, sarcomas, skin cancer, cancer of the digestive organs, andbreast cancer. 5-Fluorouracil, however, causes serious adverse reactionssuch as nausea, alopecia, diarrhea, stomatitis, leukocyticthrombocytopenia, anorexia, pigmentation, and edema. Derivatives of5-fluorouracil with anti-cancer activity have been described in U.S.Pat. No. 4,336,381,

U.S. Pat. No. 4,000,137 discloses that the peroxidate oxidation productof inosine, adenosine, or cytidine with methanol or ethanol has activityagainst lymphocytic leukemia.

Cytosine arabinoside (also referred to as Cytarabin, araC, and Cytosar)is a nucleoside analog of deoxycytidine that was first synthesized in1950 and introduced into clinical medicine in 1963. It is currently animportant drug in the treatment of acute myeloid leukemia. It is alsoactive against acute lymphocytic leukemia, and to a lesser extent, isuseful in chrome myelocytic leukemia and non-Hodgkin's lymphoma. Theprimary action of araC is inhibition of nuclear DNA synthesis.Handschumacher, R. and Cheng, Y., “Purine and PyrimidineAntimetabolites”, Cancer Medicine. Chapter XV-1, 3rd Edition, Edited by3. Holland, et al., Lea and Febigol, publishers.

5-Azacytidine is a cytidine analog that is primarily used in thetreatment of acute myelocytic leukemia and myelodysplastic syndrome.

2-Fluoroadenosine-5′-phosphate (Fludara, also referred to as FaraA)) isone of the most active agents in the treatment of chronic lymphocyticleukemia. The compound acts by inhibiting DNA synthesis. Treatment ofcells with F-araA is associated with the accumulation of cells at theG1/S phase boundary and in S phase; thus, it is a cell cycle Sphase-specific drug. Incorporation of the active metabolite, F-araATP,retards DNA chain elongation. F-araA is also a potent inhibitor ofribonucleotide reductase, the key enzyme responsible for the formationof dATP.

2-Chlorodeoxyadenosine is useful in the treatment of low grade B-cellneoplasms such as chronic lymphocytic leukemia, non-Hodgkins' lymphoma,and hairy-cell leukemia.

In designing new biologically active nucleosides, there have been anumber of attempts to incorporate a fluoro substituent into thecarbohydrate ring of the nucleoside. Fluorine has been suggested as asubstituent because it might serve as an isopolar and isosteric mimic ofa hydroxyl group as the C—F bond length (1.35 Å) is so similar to theC—O bond length (1.43 Å) and because fluorine is a hydrogen bondacceptor. Fluorine is capable of producing significant electronicchanges in a molecule with minimal steric perturbation. The substitutionof fluorine for another group in a molecule can cause changes insubstrate metabolism because of the high strength of the C—F bond (116kcal/mol vs. C—H=100 kcal/mol).

A number of references have reported the synthesis and use of2-arabinofluoro-nucleosides (i.e., nucleosides in which a 2′-fluorogroup is in the “up”-configuration). There have been several reports of2-fluoro-β-D-arabinofuranosyl nucleosides that exhibit activity againsthepatitis B and herpes. See, for example, U.S. Pat. No. 4,666,892 toFox, et al.; U.S. Pat. No. 4,211,773 to Lopez, et al; Su, et al.,Nucleosides. 136, “Synthesis and Antiviral Effects of Several1-(2-Deoxy-2-fluoro-β-D-arabinofuranosyl)-5-alkyluracils” “SomeStructure-Activity Relationships,” J. Med. Chem., 1986, 29, 151-154;Borthwick, et al, “Synthesis and Enzymatic Resolution of Carbocyclic2′-Ara-fluoro-Guanosine: A Potent Mew Anti-Herpetic Agent,” J. Chem.Soc., Chem. Commun, 1988; Wantanabe, et al., “Synthesis and Anti-HIVActivity of 2′-“Up”-Fluoro Analogues of Active Anti-Aids Nucleosides3′-Azido-3′-deoxythymidine (AZT) and 2′,3′-dideoxycytidine (DDC),” J.Med. Chem. 1990, 33, 2145-2150; Martin, et al, “Synthesis and AntiviralActivity of Monofluoro and Difluoro Analogues of PyrimidineDeoxyribonucleosides against Human immunodeficiency Virus (HIV-1),” J.Med., Chem. 1990, 33, 2137-2145; Sterzycki, et al., “Synthesis andAnti-HIV Activity of Several 2′-Fluoro-Containing PyrimidineNucleosides,” J. Med. Chem. 1990, as well as EPA 0 316 017 also filed bySterzycki, et al; and Montgomery, et al.,“9-(2-Deoxy-2-fluoro-β-D-arabinofuranosyl)guanine: A MetabolicallyStable Cytotoxic Analogue of 2′-Deoxyguanosine.” U.S. Pat. No. 5,246,924discloses a method for treating a hepatitis infection that includes theadministration of1-(2′-deoxy-2′-fluoro-β-D-arabinofuranosyl)-3-ethyluracil), alsoreferred to as “FEAU.” U.S. Pat. No. 5,034,518 discloses2-fluoro-9-(2-deoxy-2-fluoro-β-D-arabinofuranosyl)adenine nucleosideswhich exhibit anticancer activity by altering the metabolism of adeninenucleosides by reducing the ability of the compound to serve as asubstrate for adenosine. EPA 0 292 023 discloses that certainβ-D-2′-fluoroarabinonucleosides are active against viral infections.

U.S. Pat. No. 5,128,458 disclosesβ-D-2′,3′-dideoxy-4′-thioribonucleosides as antiviral agents. U.S. Pat.No. 5,446,029 discloses that 2′,3′-dideoxy-3′-fluoronucleosides haveantihepatitis activity.

European Patent Application No. 0 409 227 A2 discloses certain3′-substituted β-D-pyrimidine and purine nucleosides for the treatmentof hepatitis B.

It has also been disclosed that L-FMAU(2′-fluoro-5-methyl-β-L-arabinofuranosyluracil) is a potent anti-HBV andanti-EBV agent. See Chu, et al, “Use of2′-Fluoro-5-methyl-β-L-arabinofuranosyluracil as a Novel Antiviral Agentfor Hepatitis B Virus and Epstein-Barr Virus” Antimicrobial Agents andChemotherapy, April 1995 pages. 979-981; Balakrishna, et al.,“Inhibition of Hepatitis B Virus by a Novel L-Nucleoside,2′-Fluoro-5-Methyl-β-L-arabinofuranosyl Uracil” Antimicrobial Agents andChemotherapy, February 1996, pages 380-356; U.S. Pat. Nos. 5,587,362;5,567,688; and 5,565,438.

U.S. Pat. Nos. 5,426,183 and 5,424,416 disclose processes for preparing2′-deoxy-2′,2′-difluoronucleosides and 2′-deoxy-2′-fluoro nucleosides.See also “Kinetic Studies of 2′,2′-difluorodeoxycytidine (Gemcitabine)with Purified Human Deoxycytidine Kinase and Cytidine Deaminase;”BioChemical Pharmacology, Vol. 45 (No. 9) pages 4857-1861, 1993,

U.S. Pat. No. 5,446,029 to Eriksson, et al., discloses that certain2′,3′-dideoxy-3′-fluoronucleosides have hepatitis B activity. U.S. Pat.No. 5,128,458 discloses certain 2′,3′-dideoxy-4′-thioribonucleosideswherein the 3′-substituent is H, azide or fluoro. WO 94/14831 disclosescertain 3′-fluoro-dihydropyrimidine nucleosides. WO 92/08727 disclosesβ-L-2′-deoxy-3′-fluoro-5-substituted uridine nucleosides for thetreatment of herpes simplex 1 and 2.

EPA Publication No. 0 352 248 discloses a broad genus of L-ribofuranosylpurine nucleosides for the treatment of HIV, herpes, and hepatitis.While certain 2′-fluorinated purine nucleosides fall within the broadgenus, there is no information given in the specification on how to makethese compounds in the specification, and they are not amongspecifically disclosed or the preferred list of nucleosides in thespecification. The specification does disclose how to make3′-ribofuranosyl fluorinated nucleosides. A similar specification isfound in WO 88/09001, filed by Aktiebolaget Astra.

European Patent Application 0 357 571 discloses a broad group of β-D andα-D pyrrolidine nucleosides for the treatment of AIDS which among thebroad class generically includes nucleosides that can be substituted inthe 2′ or 3′-position with a fluorine group. Among this broad class,however, there is no specific disclosure of 2′-fluorinated nucleosidesor a method for their production.

EPA 0 463 470 discloses a process for the preparation of(5S)-3-fluoro-tetrahydro-5-[(hydroxy)methyl]-2-(3H)-furanone, a knownintermediate in the manufacture of 2′-fluoro-2′,3′-dideoxynucleosidessuch as 2′-fluoro-2′,3′-dideoxycytidine.

U.S. Ser. No. 07/556,713 discloses β-D-2′-fluoroarabinofuranosylnucleosides, and a method for their production, which axe intermediatesin the synthesis of 2′,3′-dideoxy-2′-fluoroarabinosyl nucleosides.

U.S. Pat. No. 4,625,020 discloses a method of producing1-halo-2-deoxy-2-fluoroarabinofuranosyl derivatives bearing protectiveester groups from 1,3,5-tri-O-acyl-ribofuranose.

There appears to be a lack of disclosure of β-L-2′-fluoro-ribofuranosylnucleosides for medicinal uses, including for HIV, hepatitis (B or C),or proliferative conditions. At least with respect to 2′-ribofuranosylnucleosides, this may be because of the prior perceived difficulty inplacing a fluoro group in the 2′-ribofuranosyl configuration. Withrespect to L-2′-fluoro-2′,3′-unsaturated purine nucleosides, it may bebecause the purine nucleosides are unstable in acidic media, resultingin glycosyl bond cleavage.

In light of the fact that HIV acquired immune deficiency syndrome,AIDS-related complex, and hepatitis B and C viruses have reachedepidemic levels worldwide, and have tragic effects on the infectedpatient, there remains a strong need to provide new effectivepharmaceutical agents to treat these diseases that have low toxicity tothe host. Further, there is a need to provide new antiproliferativeagents.

Therefore, it is an object of the present invention to provide a methodand composition for the treatment of human patients infected withhepatitis B or C.

It is another object of the present invention to provide a method andcomposition for the treatment of human patients infected with HIV.

It is a further object of the present invention to provide newantiproliferative agents.

It is still another object of the present invention to provide a newprocess for the preparation of 2′-fluoro-ribofuranosyl nucleosides.

It is yet another object of the present invention to provide a newprocess for the preparation of2′,3′-dideoxy-2′,3′-didehydro-2′-fluoro-L-gycero-pent-2-eno-furanosylnucleosides.

SUMMARY OF THE INVENTION

In one embodiment of the invention, a 2′-α-fluoro-nucleoside is providedof the structure:

wherein

Base is a purine or pyrimidine base as defined further herein;

R¹ is OH, H, OR³, N₃, CN, halogen, including F, or CF₃, lower alkyl,amino, lower alkylamino, di(lower)alkylamino, or alkoxy, and base refersto a purine or pyrimidine base;

R² is H, phosphate, including monophosphate, diphosphate, triphosphate,or a stabilized phosphate prodrug; acyl, or other pharmaceuticallyacceptable leaving group which when administered in vivo, is capable ofproviding a compound wherein R² is H or phosphate; sulfonate esterincluding alkyl or arylalkyl sulfonyl including methanesulfonyl, benzyl,wherein the phenyl group is optionally substituted with one or moresubstituents as described in the definition of aryl given above, alipid, including a phospholipid, an amino acid, peptide, or cholesterol;and

R³ is acyl, alkyl, phosphate, or other pharmaceutically acceptableleaving group which when administered in vivo, is capable of beingcleaved to the parent compound.

In a second embodiment, a 2′-fluoronucleoside is provided of theformula:

wherein the substituents are as defined above.

In a third embodiment a 2′-fluoronucleoside is provided of the formula:

wherein the substituents are as defined above.In a fourth embodiment, a 2′-fluoronucleoside is provided of thestructure:

wherein the substituents are as defined above.

These 2′-fluoronucleosides can be either in the β-L or β-Dconfiguration. The β-L configuration is preferred.

The 2′-fluoronucleosides are biologically active molecules which areuseful in the treatment of hepatitis B, hepatitis C or HIV. Thecompounds are also useful for the treatment of abnormal cellularproliferation. Including tumors and cancer. One can easily determine thespectrum of activity by evaluating the compound in the assays describedherein or with another confirmatory assay.

In another embodiment, for the treatment of hepatitis or HIV, the activecompound or its derivative or salt can be administered in combination oralternation with another antiviral agent, such as an anti-HIV agent oranti-hepatitis agent, including those of the formula above. In general,in combination therapy, an effective dosage of two or more agents areadministered together, whereas during alternation therapy, an effectivedosage of each agent is administered serially. The dosages will dependon absorption, inactivation, and excretion rates of the drug as well asother factors known to those of skill in the art. It is to be noted thatdosage values will also vary with the severity of the condition to bealleviated. It is to be further understood that for any particularsubject, specific dosage regimens and schedules should be adjusted overtime according to the individual need and the professional judgment ofthe person administering or supervising the administration of thecompositions.

Nonlimiting examples of antiviral agents mat can be used in combinationwith the compounds disclosed herein include2-hydroxymethyl-5-(5-fluorocytosin-1-yl)-1,3-oxathiolane (FTC); the(−)-enantiomer of 2-hydroxymethyl-5(cytosin-1-yl)-1,3-oxathiolane (3TC);carbovir, acyclovir, interferon, famciclovir, penciclovlr, AZT, DDI,DDC, D4T, abacavir, L-(−)-FMAU, L-DDA phosphate prodrugs, andβ-D-dioxolane nucleosides such as β-D-dioxolanyl-guanine (DG),β-D-dioxolanyl-2,6-diaminopurine (DAPD), andβ-D-dioxolanyl-6-chloropurine (ACP), non-nucleoside RT inhibitors suchas nevirapine, MKC-442, DMP-266 (sustiva) and also protease inhibitorssuch as indinavir, saquinavir, AZT, DMP-450 and others.

The compounds can also be used to treat equine infectious anemia virus(EIAV), feline immunodeficiency virus, and simian immunodeficiencyvirus. (Wang, S., Montelaro, R., Schinazi, R. F., Jagerski. B., andMellors, J. W.: “Activity of nucleoside and non-nucleoside reversetranscriptase inhibitors (NNRTI) against equine infectious anemia virus(EIAV)” First National Conference on Human Retro viruses and RelatedInfections, Washington, D.C., Dec. 12-16, 1993; Sellon D. C., “EquineInfectious Anemia,” Vet. Clin. North Am. Equine Pract. United States,9:321-336, 1993; Philpott, M. S., Ebner, J. P., Hoover, E. A.,“Evaluation of 9-(2-phosphonylmethoxyethyl) adenine therapy for felineimmunodeficiency virus using a quantitative polymerase chain reaction,”Vet. Immunol. Immunopathol. 35:155166, 1992.)

A new and completely diastereoselective method for the introduction offluorine into a non-carbohydrate sugar ring precursor is also provided.The method includes reacting a chiral, non-carbohydrate sugar ringprecursor (4S)-5-(protected oxy)-pentan-4-olide, which can be preparedfrom L-glutamic acid, with an electrophilic source of fluorine,including but not limited to N-fluoro-(bis)benzenesulfonimide, to yieldkey intermediate fluorolactone 6. The fluorolactone is reduced to thelactol and acetylated to give the anomeric acetate and then used for thesynthesis of a number of novel β-L-α-2′-fluoronucleosides. Thecorresponding D-enantiomer can also be synthesized using D-glutamic acidas a starting material.

In an alternative embodiment, a fluorinated glycol is prepared which isdehydrogenated and then converted to a2′,3′-dideoxy-2′,3′-didehydro-2′-fluoronucleoside or a β-L orβ-D-arabinosyl-2′-fluoronucleoside, as discussed further below.

A method for the facile preparation of2′,3′-dideoxy-2′,3′-didehydro-2′-fluoronucleosides is also presentedthat includes the direct condensation of silylated 6-chloropurine withkey immediate, which is prepared from L-2,3-0-isopropylideneglyceraldenhyde.

DETAILED DESCRIPTION OF THE INVENTION

The invention as disclosed herein is a compound, method and compositionfor the treatment of HIV, hepatitis (B or C), or abnormal cellularproliferation, in humans or other host animals, that includesadministering an effective amount of a 2′-fluoro-nucleoside, apharmaceutically acceptable derivative, including a compound which hasbeen alkylated or acylated at the 5′-position or on the purine orpyrimidine, or a pharmaceutically acceptable salt thereof, optionally ina pharmaceutically acceptable carrier. The compounds of this inventioneither possess antiviral (i.e., anti-HIV-1, anti-HIV-2, oranti-hepatitis (B or C)) activity, or antiproliferative activity, or aremetabolized to a compound that exhibits such activity.

In summary, the present invention includes the following features:

(a) β-L and β-D-2′-fluoronucleosides, as described herein, andpharmaceutically acceptable derivatives and salts thereof;

(b) β-L and β-D-2′-fluoronucleosides as described herein, andpharmaceutically acceptable derivatives and salts thereof for use inmedical therapy, for example for the treatment or prophylaxis of an HIVor hepatitis (B or C) infection or for the treatment of abnormalcellular proliferation:

(c)2′,3′-Dideoxy-2′,3′-didehydro-2′-fluoro-L-glycero-pen-2-eno-furanosylnucleosides, and pharmaceutically acceptable derivatives and saltsthereof for use in medical therapy, for example for the treatment orprophylaxis of an HIV or hepatitis (B or C) infection or for thetreatment of abnormal cellular proliferation

(d) use of these 2′-fluoronucleosides, and pharmaceutically acceptablederivatives and salts thereof in the manufacture of a medicament fortreatment of an HIV or hepatitis infection or for the treatment ofabnormal cellular proliferation;

(e) pharmaceutical formulations comprising the 2′-fluoronucleosides or apharmaceutically acceptable derivative or salt thereof together with apharmaceutically acceptable carrier or diluent;

(f) processes for the preparation of β-L and β-D-2′-α-fluoronucleosides,as described in more detail below, and

(g) processes for the preparation of2′,3′-dideoxy-2′,3′-didehydro-2′-fluoro-L-gycero-pent-2-eno-furanosylnucleosides.

I. Active Compound, and Physiologically Acceptable Derivatives and SaltsThereof

A 2′-α-fluoro-nucleoside is provided of the structure:

wherein R¹ is H, OH, OR³, N₃, CN, halogen, including F, or CF₃, loweralkyl, amino, lower alkylamino, di(lower)alkylamino, or alkoxy, and baserefers to a purine or pyrimidine base.

R² is H, phosphate, including monophosphate, diphosphate, triphosphate,or a stabilized phosphate prodrug; acyl, or other pharmaceuticallyacceptable leaving group which when administered in vivo, is capable ofproviding a compound wherein R² is H or phosphate, sulfonate esterincluding alkyl or arylalkyl sulfonyl including methanesulfonyl, benzyl,wherein the phenyl group is optionally substituted with one or moresubstituents as described in the definition of aryl given above, alipid, an amino acid, peptide, or cholesterol; and

R³ is acyl, alkyl, phosphate, or other pharmaceutically acceptableleaving group which when administered in vivo, is capable of beingcleaved to the parent compound.

In a second embodiment, a 2-fluoronucleoside is provided of the formula:

In a third embodiment, a 2-fluoronucleoside is provided of the formula:

in a fourth embodiment, a 2-fluoronucleoside is provided of thestructure:

The term alkyl, as used herein, unless otherwise, specified, refers to asaturated straight, branched, or cyclic, primary, secondary, or tertiaryhydrocarbon of C₁ to C₁₀, and specifically includes methyl, ethyl,propyl, isopropyl, cyclopropyl, butyl, isobutyl, t-butyl pentyl,cyclopentyl, isopentyl, neopentyl, hexyl, isohexyl, cyclohexyl,cyclohexylmethyl, 3-methylpentyl,2,2-dimethylbutyl and2,3-dimethylbutyl. The alkyl group can be optionally substituted withone or more moieties selected from the group consisting of hydroxyl,amino, alkylamino, arylamino, alkoxy, aryloxy, nitro, cyano, sulfonicacid, sulfate, phosphonic acid, phosphate, or phosphonate, eitherunprotected, or protected as necessary, as known to those skilled in theart, for example, as taught in Greene, et ah, Protective Groups inOrganic Synthesis, John Wiley and Sons, Second Edition, 1991, herebyincorporated by reference.

The term lower alkyl, as used herein, and unless otherwise specified,refers to a C₁ to C₄ saturated straight, branched, or if appropriate, acyclic (for example, cyclopropyl)alkyl group.

The term alkylamino or arylamino refers to an amino group that has oneor two alkyl or aryl substituents, respectively.

The term “protected” as used herein and unless otherwise defined refersto a group that is added to an oxygen, nitrogen, or phosphorus atom toprevent its further reaction or for other purposes. A wide variety ofoxygen and nitrogen protecting groups are known to those skilled in theart of organic synthesis. The term aryl, as used herein, and unlessotherwise specified, refers to phenyl, biphenyl, or naphthyl, andpreferably phenyl. The aryl group can be optionally substituted with oneor more moieties selected from the group consisting of hydroxyl, amino,alkylamino, arylamino, alkoxy, aryloxy, nitro, cyano, sulfonic acid,sulfate, phosphoric acid, phosphate, or phosphonate, either unprotected,or protected as necessary, as known to those skilled in the art, forexample, as taught in Greene, et al., Protective Groups in OrganicSynthesis, John Wiley and Sons, Second Edition, 1991.

The term alkaryl or alkylaryl refers to an alkyl group with an arylsubstituent. The term aralkyl or arylalkyl refers to an aryl group withan alkyl substituent.

The term halo, as used herein, includes chloro, bromo, iodo, and fluoro.

The term purine or pyrimidine base includes, but is not limited to,adenine, N⁶-alkylpurines, N⁶-acylpurines (wherein acyl is C(O)(alkyl,aryl, alkylaryl, or arylalkyl), N⁶-benzylpurine, N⁶-halopurine,N⁶-vinylpurine, N⁶-acetylenic purine, N⁶-acyl purine, N⁶-hydroxyalkylpurine, N⁶-thioalkyl purine, N²-alkylpurines, N⁶-alkyd-6-thiopurines,thymine, cytosine, 5-fluorocytosine, 5-methylcytosine, 6-azapyrimidine,including 6-azacytosine, 2- and/or 4-mercaptopyrmidine, uracil,5-halouracil, including 5-fluorouracil, C⁵-alkylpyrimidines,C⁵-benzylpyrimidines, C⁵-halopyrimidines, C⁵-vinylpyrimidine,C⁵-acetylenic pyrimidine, C⁵-acyl pyrimidine, C⁵-hydroxyalkyl purine,C⁵-amidopyrimidine, C⁵-cyanopyrimidine, C⁵-nitropyrimidine,C⁵-aminopyrimidine, N²-alkylpurines, N²-alkyl-6-thiopurines,5-azacytidinyl, 5-azauracilyl, triazolopyridinyl, imidazolopyridinyl,pyrrolopyrimidinyl, and pyrazolopyrimidinyl. Purine bases include, butare not limited to, guanine, adenine, hypoxanthine, 2,6-diaminopurine,and 6-chloropurine. Functional oxygen and nitrogen groups on the basecan be protected as necessary or desired. Suitable protecting groups arewell known to those skilled in the art, and include trimethylsilyl,dimethylhexylsiiyl, t-butyldimethylsilyl, and t-butyldiphenylsilyl,trityl, alkyl groups, acyl groups such as acetyl and propionyl,methanesulfonyl, and p-toluenesulfonyl.

The active compound can be administered as any derivative that uponadministration to the recipient, is capable of providing directly orindirectly, the parent compound, or that exhibits activity itself.Nonlimiting examples are the pharmaceutically acceptable salts(alternatively referred to as “physiologically acceptable, salts”), anda compound which has been alkylated or acylated at the 5′-position or onthe purine or pyrimidine base (alternatively referred to as“pharmaceutically acceptable derivatives”). Further, the modificationscan affect the biological activity of the compound, in some casesincreasing the activity over the parent compound. This can easily beassessed by preparing the derivative and testing its antiviral activityaccording to the methods described herein, or other method known tothose skilled in the art.

The term acyl refers to a carboxylic acid ester in which thenon-carbonyl moiety of the ester group is selected from straight,branched, or cyclic alkyl or lower alkyl, alkoxyalkyl includingmethoxymethyl, aralkyl including benzyl, aryloxyalkyl such asphenoxymethyl, aryl including phenyl optionally substituted withhalogen, C₁ to C₄ alkyl or C₁ to C₄ alkoxy, sulfonate esters such asalkyl or aralkyl sulphonyl including methanesulfonyl, the mono, di ortriphosphate ester, trityl or monomethoxytrityl, substituted benzyl,trialkylsilyl (e.g. dimethyl-t-butylsilyl) or diphenylmethylsilyl. Arylgroups in the esters optimally comprise a phenyl group.

As used herein, the term “substantially free of” or “substantially inthe absence of” refers to a nucleoside composition that includes atleast 95% to 98%, or more preferably, 99% to 100%, of the designatedenantiomer of that nucleoside.

Nucleotide Prodrug Formulations

Any of the nucleosides described herein can be administrated as anucleotide prodrug to increase the activity, bioavailability, stabilityor otherwise alter the properties of the nucleoside. A number ofnucleotide prodrug ligands are known. In general, alkylation, acylationor other lipophilic modification of the mono, di or triphosphate of thenucleoside will increase the stability of the nucleotide. Examples ofsubstituent groups that can replace one or more hydrogens on thephosphate moiety are alkyl, aryl, steroids, carbohydrates, includingsugars, 1,2-diacylglycerol and alcohols. Many are described in R. Jonesand N. Bischofberger, Antiviral Research, 27 (1995) 1-17. Any of thesecan be used in combination with the disclosed nucleosides to achieve adesired effect.

The active nucleoside can also be provided as a 5′-phosphoether lipid ora 5′-ether lipid, as disclosed in the following references, which areincorporated by reference herein: Kucera, L. S., N. Iyer, E. Leake, A.Raben, Modest E. K., D. L. W., and C. Piantadosi. 1990. “Novelmembrane-interactive ether lipid analogs that inhibit infectious HIV-1production and induce defective virus formation.” AIDS Res. Hum. RetroViruses. 6:491-501; Piantadosl, C., J. Marasco C. J., S. L.Morris-Natschke, K. L. Meyer, F. Gumus, J. R. Surles, K. S. Ishaq. L. S.Kucera, N. Iyer, C. A. Wallen, S. Piantadosi, and E. J. Modest. 1991.“Synthesis and evaluation of novel ether lipid nucleoside conjugates foranti-HIV activity.” J. Med. Chem. 34:1408.1414; Hosteller, K. Y., D. D.Richman, D. A. Carson, L. M. Stuhmiller, G. M. T. van Wijk, and H. vanden Bosch. 1992. “Greatly enhanced inhibition of human immunodeficiencyvirus type 1 replication in CEM and HT4-6C cells by 3′-deoxythymidinediphosphate dimyristoylglycerol, a lipid prodrug of 3,-deoxthymidine.”Antimicrob. Agents Chemother. 36:2025.2029; Hosetler, K. Y., L. M.Stuhmiller, H. B. Lenting, H. van den Bosch, and D. D. Richman, 1990.“Synthesis and antiretroviral activity of phospholipid analogs ofazidothymidine and other antiviral nucleosides.” J. Biol. Chem.265:61127.

Nonlimiting examples of U.S. patents that disclose suitable lipophilicsubstituents that can be covalently incorporated into the nucleoside,preferably at the 55-OH position of the nucleoside or lipophilicpreparations, include U.S. Pat. No. 5,149,794 (Sep. 22, 1992, Yatvin etal.); U.S. Pat. No 5,194,654 (Mar. 16, 1993, Hostetler et ah, U.S. Pat.No 5,223,263 (Jun. 29, 1993, Hostetler et al.); U.S. Pat. No 5,256,641(Oct. 26, 1993, Yatvin et al.); 5,411,947 (May 2, 1995, Hostetler etal.); U.S. Pat. No 5,463,092 (Oct. 31, 1995, Hostetler et al.); U.S.Pat. No 5,543,389 (Aug. 6, 1996, Yatvin et al.); U.S. Pat. No 5,543,390(Aug. 6, 1996, Yatvin et al.); U.S. Pat. No 5,543,391 (Aug. 6, 1996,Yatvin et al.); and 5,554,728 (Sep. 10, 1996; Basava et al.), all ofwhich are incorporated herein by reference. Foreign patent applicationsthat disclose lipophilic substituents that can be attached to thenucleosides of the present invention, or lipophilic preparations,include WO 89/02733, WO 90/00555, WO 91/16920, WO 91/18914, WO 93/00910,WO 94/26273, WO 96/15132, EP 0 350 287, EP 93917054.4, and WO 91/19721.

Nonlimiting examples of nucleotide prodrugs are described in thefollowing references: Ho, D. H. W. (1973) “Distribution of Kinase anddeaminase of 1β-D-arabinofuranosylcytosine in tissues of man and muse.”Cancer Res. 33, 2816-2820; Holy, A. (1993) Isopolar phosphorous-modifiednucleotide analogues,” In: De Clercq (Ed.), Advances in Antiviral DrugDesign, Vol. I, JAI Press, pp. 179-231; Hong, C. L, Nechaev, A., andWest, C. R. (1979a) “Synthesis and antitumor activity of1-β-D-arabino-furanosylcytosine conjugates of Cortisol and cortisone.”Bicohem. Biophys. Rs. Commun. 88, 1223-1229; Hong, C. L, Nechaev, A.,Kirisits, A. J. Buchheit, D. J. and West, C. R. (1980) “Nucleosideconjugates as potential antitumor agents. 3. Synthesis and antitumoractivity of 1-(β-D-arabinofuranosyl) cytosine conjugates ofcorticosteriods and selected lipophilic alcohols.” J. Med. Chem. 28,171-177; Hosteller, K. Y., Stuhmiller, L. M., Lenting, H. B. M. van denBosch, H. and Richman J. Biol. Chem. 265, 6112-6117; Hosteller, K. Y.,Carson, D. A. and Richman, D. D. (1991); “Phosphatidylazidothymidine:mechanism of antiretroviral action in CEM cells.” J. Biol. Chem. 266,11714-11717; Hosteller, K. Y., Korba, B. Sridhar, C., Gardener, M.(1994a) “Antiviral activity of phosphatidyl-dideoxycytidine in hepatitisB-infected cells and enhanced hepatic uptake in mice.” Antiviral Res.24, 59-67; Hosteller, K. Y., Richman, D. D., Sridhar. C. N. Felgner, P.L. Felgner, J., Ricci, J., Gardener, M. F. Selleseth, D. W. and Ellis,M. N. (1994b) “Phosphatidylazidothymidine and phosphatidyl-ddC:Assessment of uptake in mouse lymphoid tissues and antiviral activitiesin human immunodeficiency virus-infected cells and in rauscher leukemiavirus-infected mice.” Antimicrobial Agents Chemother. 38, 2792-2797;Hunston, R. N., Jones, A. A. McGuigan, C., Walker, R. T., Balzarini, J.,and DeClercq, E. (1984) “Synthesis and biological properties of somecyclic phosphotriesters derived from 2′-deoxy-5-fluorouridine.” J. Med.Chem. 21, 440-444; Ji, Y. H., Moog, C., Schmitt, G., Bischoff, P. andLuu, B. (1990); “Monophosphoric acid esters of 7-β-hydroxycholesteroland of pyrimidine nucleoside as potential antitumor agents: synthesisand preliminary evaluation of antitumor activity.” J. Med. Chem. 332264-2270; Jones, A. S., McGuigan, C., Walker, R. T., Balzarini, J. andDeClercq, E. (1984) “Synthesis, properties, and biological activity ofsome nucleoside cyclic phosphoramidates.” J. Chem. Soc. Perkin Trans, I,1471-1474; Juodka, B. A. and Smrt, J. (1974) “Synthesis ofdiribonucleoside phosph (P→N) amino acid derivatives.” Coll. Czech.Chem. Comm. 39, 363-968; Kataoka, S., Imai, J., Yamaji, N. Kato, M.,Saito, M., Kawada, T. and Imai, S. (1989) “Alkylated cAMP derivatives;selective synthesis and biological activities.” Nucleic Acids Res. Sym.Ser. 21, 1-2; Kataoka, S., Uchida, “(cAMP) benzyl and methyl triesters.”Heterocycles 32, 1351-1356; Kinchington, D., Harvey, J. J., O'Connor, T.J., Jones, B. C. N. M., Devine, K. G., Taylor-Robinson D., Jeffries, D.J. and McGuigan, C. (1992) “Comparison of antiviral effects ofzidovudine phosphoramidate an diphosphorodiamidate derivates against HIVand ULV in vitro.” Antiviral Chem. Chemother. 3, 107-112; Kodama, K.,Morozumi, M., Saithoh, K. I., Kuninaka, H., Yosino, H. and Saneyoshi, M.(1989) “Antitumor activity and pharmacology of1-β-D-arabinofuranosylcytosine -5′-stearylphosphate; an orally activederivative of 1-β-D-arabinofuranosylcytosine.” Jpn. J. Cancer Res. 80,679-685; Korty, M. and Engels, J. (1979) “The effects of adenosine- andguanosine 3′,5′phosphoric and acid benzyl esters on guinea-pigventricular myocardium.” Naunyn-Schmiedeberg's Arch. Pharmacol. 310,103-111; Kumar, A., Goe, P. L., Jones, A. S. Walker, R. T. Balzarini, J.and DeClercq, E. (1990) “Synthesis and biological evaluation of somecyclic phosphoramidate nucleoside derivatives.” J. Med. Chem., 33,2368-2375; LeBec, C., and Huynh-Dinh, T. (1991) “Synthesis of lipophilicphosphate triester derivatives of 5-fluorouridine an arabinocytidine asanticancer prodrugs.” Tetrahedron Lett. 32, 6553-6556; Lichtenstein, J.,Barner, H. D. and Cohen, S. S. (1960) “The metabolism of exogenouslysupplied nucleotides by Escherichia coli.,” J. Biol. Chem. 235, 457-465;Lucthy, J., Von Daeniken, A., Friederich, J. Manthey, B., Zweifel, J.,Schlatter, C. and Benn, M. H. (1981) “Synthesis and toxicologicalproperties of three naturally occurring cyanoepithioalkanes”. Mitt. Geg.Lehensmittelunters. Hyg. 72, 131-133 (Chem. Abstr. 95, 127093); McGigan,C. Tollerfield, S. M. and Riley, P. a. (1989) “Synthesis and biologicalevaluation of some phosphate triester derivatives of the anti-viral drugAra.” Nucleic Acids Res. 17, 6065-6075; McGuigan, C., Devine, K. G.,O'Connor, T. J., Galpin, S. A., Jeffries, D. J. and Kinchington, D.(1990a) “Synthesis and evaluation of some novel phosphoramidatederivatives of 3′-azido-3′-deoxythymidine (AZT) as anti-HIV compounds.”Antiviral Chem. Chemother. 1 107-113; McGuigan, C., O'Connor, T. J.,Nicholls, S. R. Nickson, C. and Kinchington, D. (1990b) “Synthesis andanti-HIV activity of some novel substituted dialkyl phosphatederivatives of AZT and ddCyd.” Antiviral Chem. Chemother. 1, 355-360;McGuigan, C., Nicholls, S. R., O'Connor, T. J., and Kinchington, D.(1990c) “Synthesis of some novel dialkyl phosphate derivative of3′-modified nucleosides as potential anti-AIDS drugs.” Antiviral Chem.Chemother. 1, 25-33; McGuigan, C., Devin, K. G., O'Connor, T. J., andKinchington, D. (1991) “Synthesis and anti-HIV activity of somehaloalkyl phosphoramidate derivatives of 3′-azido-3′-deoxy thymidine(AZT); potent activity of the trichloroethyl methoxyalaninyl compound.”Antiviral Res. 15, 255-263; McGuigan, C, Pathirana, R. N., Balzarini, J.and DeClercq, E. (1993b) “Intracellular delivery of bioactive AZTnucleotides by aryl phosphate derivatives of AZT.” J. Med. Chem. 36,1048-1052.

Alkyl hydrogen phosphate derivatives of the anti-HIV agent AZT may beless toxic than the parent nucleoside analogue. Antiviral Chem.Chemother. 5, 271-277; Meyer. R. B., Jr., Shaman, D. A. and Robins, R.K. (1973) “Synthesis of purine nucleoside 3′,5′-cyclicphosphoramidates.” Tetrahedron Lett. 269-272; Nagyvary, J. Gohil, R. N.,Kirchner, C. R. and Stevens, J. D. (1973) “Studies on neutral esters ofcyclic AMP,” Biochem. Biophys. Res. Commun. 55, 1072-1077; Namane, A.Gouyette, C., Fillion, M. P., Fillion, G. and Huynh-Dinh, T. (1992)“Improved brain delivery of AZT using a glycosyl phosphotriesterprodrug.” J, Med. Chem. 35, 3039-3044; Nargeot, J, Nerbonne, J. M.Engels, J. and Leser, H. A. (1983) Natl. Acad. Sci. U.S.A. 80,2395-2399; Nelson, K. A., Bentrude, W. G. Stser, W. N. and Hutchinson,J. P. (1987) “The question of chair-twist equilibria for tire phosphaterings of nucleoside cyclic 3′, 5′ monophosphates. ¹HNMR and x-raycrystallographic study of the diastereomers of thymidine phenyl cyclic3′,5′-monophosphate.” J. Am. Chem. Soc. 109, 4058-4064; Nerbonne, J. M.,Richard, S., Nargeot, J. and Lester, H. A. (1984) “New photoactivatablecyclic nucleotides produce intracellular jumps in cyclic AMP and cyclicGMP concentrations.” Nature 301, 74-76; Neumann, J. M., Herv_, M.,Debouzy, J. C., Guerra, F. C., Gouyette, C., Dupraz, B. and Huyny-Dinh,T. (1989) “Synthesis and transmembrane transport studies by NMR of aglucosyl phospholipid of thymidine.” J. Am. Chem. Soc. 111, 4270-4277;Ohno, R., Tatsumi, N., Hirano, M., Imai, K. Mizoguchi, H., Nakamura, T.,Kosaka, M., Takatuski, K., Yamaya, T., Toyama K., Yoshida, T., Masaoka,T., Hashimoto, S., Ohshima, T., Kimura, I., Yamada, K. and Kimura, J.(1991) “Treatment of myelodysplastic syndromes with orally administered1-β-D-arabinouranosylcytosine-5′ stearylphosphate.” Oncology 48,451-455. Palomino, E., Kessle, D. and Horwitz, J. P. (1989) “Adihydropyridine carrier system for sustained delivery of 2′,3′dideoxynucleosides to the brain.” J. Med. Chem. 32, 22-625; Perkins, R.M., Barney, S. Wittrock, R., Clark, P. H., Levin, R. Lambert, D. M.,Petteway, S. R., Serafinowska, H. T., Bailey, S. M., Jackson, S.,Harnden, M. R. Ashton, R., Sutton, D., Harvey, J. J. and Brown, A. G.(1993) “Activity of BRL47923 and its oral prodrug, SB203657A against arauscher murine leukemia virus infection in mice.” Antiviral Res. 20(Suppl. I). 84; Piantadosi, C., Marasco, C. J., Jr., Norris-Natschke, S.L., Meyer, K. L., Gumus, F., Surles, J. R., Ishaq, K. S., Kucera, L. S.Iyer, N., Wallen, C. A., Piantadosi, S. and Modest, E. J. (1991)“Synthesis and evaluation of novel ether lipid nucleoside conjugates foranti-HIV-1 activity.” J. Med. Chem. 34, 1408-1414; Pompon, A., Lefebvre,I., Imbach, J. L., Kahn, S. and Farquhar, D. (1994). “Decompositionpathways of the mono- and bis(pivaloyloxymethyl) esters ofazidothymidine-5′-monophosphate in cell extract and in tissue culturemedium; an application of the ‘on-line ISRP-cleaning HPLC technique.”Antiviral Chem Chemother. 5, 91-98; Postemark, T. (1974) “Cyclic AMP andcyclic GMP.” Annu. Rev. Pharmacol. 14, 23-33; Prisbe, E. J., Martin, J.C. M., McGhee, D. P. C, Barker, M. F., Smee, D. F. Duke, A. E.,Matthews, T. R. and Verheyden, J. P. J. (1986) “Synthesis and antiherpesvirus activity of phosphate an phosphonate derivatives of9-[(1,3-dihydroxy-2-propoxy)methyl]guanine.” J. Med. Chem. 29, 671-675;Pucch, F., Gosselin, G., Lefebvre, I., Pompon, a., Aubertin, A. M. Dirn,and Imbach, J. L. (1993) “Intracellular deliver of nucleosidemonophosphate through a reductase-mediated activation process.” AntivralRes. 22, 155-174; Pugaeva, V. P., Klochkeva, S. I., Mashbits, F. D. andEizengart, R. S. (1969). “Toxicological assessment and health standardratings for ethylene sulfide in the industrial atmosphere.” Gig. Trf.Prof. Zabol. 14, 47-48 (Chem. Abstr. 72, 212); Robins, R. K. (1984) “Thepotential of nucleotide analogs as inhibitors of Retro viruses andtumors.” Pharm. Res. 11-18; Rosowsky, A., Kim. S. H., Ross and J. Wick,M. M. (1982) “Lipophilic 5′-(alkylphosphate) esters of1-β-D-arabinofuranosylcytosine and its N⁴-acyl and 2.2′-anhydro-3′0-acylderivatives as potential prodrugs.” J. Med. Chem. 25, 171-178; Ross, W.(1961) “Increased sensitivity of the walker turnout towards aromaticnitrogen mustards carrying basic side chains following glucosepretreatment.” Biochem. Pharm. 8, 235-240; Ryu, E. K., Ross, R. J.Matsushita, T., MacCoss, M., Hong, C. I. and West, C. R. (1982).“Phospholipid-nucleoside conjugates. 3. Synthesis and preliminarybiological evaluation of 1-β-D-arabinofuranosylcytosine 5′ diphosphate[−], 2-diacylglycerols.” J. Med. Chem. 25, 1322-1329; Saffhill, R. andHume, W. J. (1986) “The degradation of 5-iododeoxyuridine and5-bromoethoxyuridine by serum from different sources and itsconsequences for the use of these compounds for incorporation into DNA.”Chem. Biol. Interact. 57, 347-355; Saneyoshi, M., Morozumi, M., Kodama,K., Machida, J., Kuninaka, A. and Yoshino, H. (1980) “Syntheticnucleosides and nucleotides. XVI. Synthesis and biological evaluationsof a series of 1-β-D-arabinofuranosylcytosine 5′-alky orarylphosphates.” Chem Pharm. Bull. 28, 2915-2923; Sastry, J. K., Nehete,P. N., Khan, S., Nowak, B. J., Phunkett W., Arlinghaus, R. B. andFarquhar, D. (1992) “Membrane-permeable dideoxyuridine 5′-monophosphateanalogue inhibits human immunodeficiency virus infection.” Mol.Pharmacol. 41, 441-445; Shaw, J. P., Jones, R. J. Arimilli, M. N.,Louie, M. S., Lee, W. A. and Cundy, K. C. (1994) “Oral bioavailabilityof PMEA from PMEA prodrugs in male Sprague-Dawley rats.” 9th Annual AAPSMeeting. San Diego, Calif. (Abstract). Shuto, S., Ueda, S., Imamura, S.,Fukukawa, K. Matsuda, A. and Ueda, T. (1987) “A facile one-stepsynthesis of 5′phosphatidylnucleosides by an enzymatic two-phasereaction.” Tetrahedron Lett. 28, 199-202; Shuto, S. Itoh, H., Ueda, S.,Imamura, S., Kukukawa, K., Tsujino, M., Matsuda, A. and Ueda, T. (1988)Pharm. Bull. 36, 209-217. An example of a useful phosphate prodrug groupis the S-acyl-2-thioethyl group, also referred to as “SATE”.

II. Combination and Alternation Therapy

It has been recognized that drug-resistant variants of HIV and HBV canemerge after prolonged treatment with an antiviral agent. Drugresistance most typically occurs by mutation of a gene that encodes foran enzyme used in viral replication, and most typically in the case ofHIV, reverse transcriptase, protease, or DNA polymerase, and in the caseof HBV, DNA polymerase. Recently, it has been demonstrated that theefficacy of a drug against HIV infection can be prolonged, augmented, orrestored by administering the compound in combination or alternationwith a second, and perhaps third, antiviral compound that induces adifferent mutation from that caused by the principle drug.Alternatively, the pharmacokinetics, biodistribution, or other parameterof the drug can be altered by such combination or alternation therapy.In general, combination therapy is typically preferred over alternationtherapy because it induces multiple simultaneous stresses on the virus.

The second antiviral agent for the treatment of HIV, in one embodiment,can be a reverse transcriptase inhibitor (a “RTI”), which can be eithera synthetic nucleoside (a “NRTI”) or a non-nucleoside compound (a“NNRTI”). In an alternative embodiment, in the case of HIV, the second(or third) antiviral agent can be a protease inhibitor. In otherembodiments, the second (or third) compound can be a pyrophosphateanalog, or a fusion binding inhibitor A list compiling resistance datacollected in vitro and in vivo for a number of antiviral compounds isfound in Schinazi, et al, Mutations in retroviral genes associated withdrug resistance, International Antiviral News, 1997.

Preferred compounds for combination or alternation therapy for thetreatment of HBV include 3TC, FTC, L-FMAU, interferon,β-D-dioxolanyl-guanine (DXG), β-D-dioxolanyl-2,6-diaminopurine (DAPD),and β-D-dioxolanyl-6-chloropurine (ACP), famciclovir, penciclovir,BMS-200475, bis pom PMEA (adefovir, dipivoxil); lobucavir, ganciclovir,and ribavarin.

Preferred examples of antiviral agents that can be used in combinationor alternation with the compounds disclosed herein for HIV therapyinclude cis-2-hydroxymethyl-5-(5-fluorocytosin-1-yl)-1,3-oxathiolane(FTC); the (−)-enantiomer of2-hydroxymethyl-5-(cytosin-1-yl)-1,3-oxathiolane (3TC); carbovir,acyclovir, foscarnet, interferon, AZT, DDI, DDC, D4T, CS-87(3′-azido-2′,3′-dideoxy-uridine), and β-D-dioxolane nucleosides such asβ-D-dioxolanyl-guanine (DXG), β-D-dioxolanyl-2,6-diaminopurine (DAPD),and β-D-dioxolanyl-6-chloropurine (ACP), MKC-442(6-benzyl-1-(ethoxymethyl)-5-isopropyl uracil.

Preferred protease inhibitors include crixivan (Merck), nelfinavir(Agouron), ritonavir (Abbott), saquinavir (Roche), DMP-266 (Sustiva) andDMP-450 (DuPont Merck).

A more comprehensive list of compounds that can be administered incombination or alternation with any of the disclosed nucleosides include(1S,4R)-4-[2-amino-6-cyclopropyl-amino)-9H-purin-9-yl]-2-cyclopentene-1-methanolsuccinate (“1592”, a carbovir analog; Glaxo Wellcome); 3TC:(−)-β-L-2′,3′-dideoxy-3′-thiacytidine (Glaxo Wellcome); a-APA R18893:a-nitro-anilino-phenylacetamide; A-77003; C2 symmetry-based proteaseinhibitor (Abbott); A-75925: C2 symmetry-based protease inhibitor(Abbott); AAP-BHAP: bisheteroarylpiperazine analog (Upjohn); ABT-538: C2symmetry-based protease inhibitor (Abbott);AzddU:3′-azido-2′,3′-dideoxyuridine; AZT: 3′-azido-3′-deoxythymidine(Glaxo Wellcome); AZT-p-ddI:3′-azido-3′-deoxythymidilyl-(5′,5′)-2′,3′-dideoxyinosinic acid (Ivax);BHAP: bisheteroarylpiperazine; BILA 1906:N-{1S-[[[3-[2S-{(1,1-dimethylethyl)amino]carbonyl}-4R-]3-pyridinylmethyl)thio]-1-piperidinyl]-2R-hydroxy-1S-(phenylmethyl)propyl]amino]carbonyl]-2-methylpropyl}-2-quinolinecarboxamide(Bio Mega/Boehringer-Ingelheim); BILA 2185:N-(1,1-dimethylethyl)-1-[2S-[[2-2,6-dimethyphenoxy)-1-oxoethyl]amino]-2R-hydroxy-4-phenylbutyl]4R-pyridinylthio)-2-piperidine-carboxamide(BioMega/Boehringer-Ingelheim); BM+51.0836: thiazolo-isoindolinonederivative; BMS 186, 318: aminodiol derivative HIV-1 protease inhibitor(Bristol-Myers-Squibb); d4API:9-[2,5-dihydro-5-(phosphonomethoxy)-2-furanel]adenine (Gilead); d4C:2′,3′-didehydro-2′,3′-dideoxycytidine; d4T:2′,3′-didehydro-3′-deoxythymidine (Bristol-Myers-Squibb); ddC;2′,3′-dideoxycytidine (Roche); ddI: 2′,3′-dideoxyinosine(Bristol-Myers-Squibb); DMP-266: a 1,4-dihydro-2H -3,1-benzoxazin-2-one;DMP-450:{[4R-(4-a,5-a,6-b,7-b)]-hexahydro-5,6-bis(hydroxy)-1,3-bis(3-amino)phenyl]methyl)-4,7-bis(phenylmethyl)-2H-1,3-diazepin-2-one}bismesylate(Avid); DXG:(−)-β-D-dioxolane-guanosine (Triangle);EBU-dM:5-ethyl-1-ethoxymethyl-6-(3,5-dimethylbenzyl)uracil; E-EBU:5-ethyl-1-ethoxymethyl-6-benzyluracil; DS: dextran sulfate;E-EPSeU:1-(ethoxymethyl)-(6-phenylselenyl)-5-ethyluracil; E-EPU:1-(ethoxymethyl)-(6-phenyl-thio)-5-ethyluracil;FTC:β-2′,3′-dideoxy-5-fluoro-3′-thiacytidine (Triangle);HBY097:S-4-isopropoxycarbonyl-6-methoxy-3-(methylthio-methyl)-3,4-dihydroquinoxalin-2(1H)-thione;HEPT: 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymine; HIV-1:humanimmunodeficiency virus type 1; JM2763:1,1′-(1,3-propanediyl)-bis-1,4,8,11-tetraazacyclotetradecane (JohnsonMatthey);JM3100:1,1′-[1,4-phenylenebis-(methylene)]-bis-1,4,8,11-tetraazacyclotetradecane(Johnson Matthey); KNI-272: (2S,3S)-3-amino-2-hydroxy-4-phenylbutyricacid-containing tripeptide; L-697,593;5-ethyl-6-methyl-3-(2-phthalimido-ethyl)pyridin-2(1H)-one;L-735,524:hydroxy-aminopentane amide HIV-1 protease inhibitor (Merck);L-697,661:3-{[(-4,7-dichloro-1,3-benzoxazol-2-yl)methyl]amino}-5-ethyl-6-methylpyridin-2(1H)-one; L-FDDC; (−)-β-L-5-fluoro-2′,3′-dideoxycytidine;L-FDOC:(−)-β-L-5-fluoro-dioxolane cytosine;MKC442:6-benzyl-1-ethoxymethyl-5-isopropyluracil (I-EBU;Triangle/Mitsubishi); Nevirapine:11-cyclopropyl-5,11-dihydro-4-methyl-6H-dipyridol[3,2-b:2′,3′-e]diazepin-6-one(Boehringer-Ingelheim);NSC648400:1-benzyloxymethyl-5-ethyl-6-(alpha-pyridylthio)uracil(E-BPTU); P9941: [2-pyridylacetyl-IlePheAla-y(CHOH)]2 (Dupont Merck);PFA: phosphonoformate (foscarnet; Astra); PMEA:9-(2-phosphonylmethoxyethyl)adenine (Gilead); PMPA:(R)-9-(2-phosphonyl-methoxypropyl)adenine (Gilead); Ro 31-8959:hydroxyethylamine derivative HIV-1 protease inhibitor (Roche); RPI-312:peptidyl protease inhibitor,1-[(3s)-3-(n-alpha-benzyloxycarbonyl)-1-asparginyl)-amino-2-hydroxy-4-phenylbutyryl]-n-tert-butyl-1-prolineamide;2720:6-chloro-3,3-dimethyl-4-(isopropenyloxycarbonyl)-3,4-dihydro-quinoxalin-2(1H)thione;SC-52151: hydroxyethylurea isostere protease inhibitor (Searle);SC-55389A: hydroxyethyl-urea isostere protease inhibitor (Searle); TIBOR82150:(+)-(5S)-4,5,6,7-tetrahydro-5-methyl-6-(3-methyl-2-butenyl)imidazo[4,5,1-jk][1,4]-benzodiazepin-2(1H)-thione(Janssen); TIBO 82913:(+)-(5S)-4,5,6,7-tetrahydro-9-chloro-5-methyl-6-(3-methyl-2-butenyl)imidazo[4,5,1jk]-[1,4]benzo-diazepin-2(1H)-thione(Janssen);TSAO-m3T:[2′,5′-bis-O-(tert-butyldimethylsilyl)-3′-spiro-5′-(4′-amino-1′,2′-oxathiole-2′,2′-dioxide)]-b-D-pentofuranosyl-N3-methylthymine;U90152:1-[3-[(1-methylethyl)-amino]-2-pyridinyl]-4-[[5-[(methylsulphonyl)-amino]-1H-indol-2yl]carbonyl]-piperazine;UC: thiocarboxanilide derivatives (Uniroyal);UC-781=N-[4-chloro-3-(3-methyl-2-butenyloxy)phenyl]-2-methyl-3-furancarbothioamide;UC-82=N-[4-chloro-3-(3-methyl-2-butenyloxy)phenyl]-2-methyl-3-thiophenecarbothioamide;VB 11,328: hydroxyethyl-sulphonamide protease inhibitor (Vertex);VX-478:hydroxyethylsulphonamide protease inhibitor (Vertex); XM 323:cyclic urea protease inhibitor (Dupont Merck).

Combination Therapy for the Treatment of Proliferative Conditions

In another embodiment, the compounds, when used as an antiproliferative,can be administered in combination with another compound that increasesthe effectiveness of the therapy, including but not limited to anantifolate, a 5-fluoropyrimidine (including 5-fluorouracil), a cytidineanalogue such as β-1,3-dioxolanyl cytidine or β-L-1,3-dioxolanyl5-fluorocytidine, antimetabolites (including purine antimetabolites,cytarabine, fudarabine, floxuridine, 6-mercaptopurine, methotrexate, and6-thioguanine), hydroxyurea, mitotic inhibitors (including CPT-11,Etoposide (VP-21), taxol, and vinca alkaloids such as vincristine andvinblastine, an alkylating agent (including but not limited to busulfan,chlorambucil, cyclophosphamide, ifofamide, mechlorethamine, melphalan,and thiotepa), nonclassical alkylating agents, platinum containingcompounds, bleomycin, an anti-tumor antibiotic, an anthracycline such asdoxorubicin and dannomycin, an anthracenedione, topoisomerase IIinhibitors, hormonal agents (including but not limited tocorticosteroids (dexamethasone, prednisone, and methylprednisone),androgens such as fluoxymesterone and methyltestosterone, estrogens suchas diethylstilbesterol, antiestrogens such as tamoxifen, LHRH analoguessuch as leuprolide, antiandrogens such as flutamide, aminoglutethimide,megestrol acetate, and medroxyprogesterone), asparaginase, carmustine,lomustine, hexamethyl-melamine, dacarbazine, mitotane, streptozocin,cisplatin, carboplatin, levamasole, and leucovorin. The compounds of thepresent invention can also be used in combination with enzyme therapyagents and immune system modulators such as an interferon, interleukin,tumor necrosis factor, macrophage colony-stimulating factor and colonystimulating factor.

III. Process for the Preparation of Active Compounds

In one embodiment of the invention, a diastereoselective reaction foreffecting the introduction of fluorine into the sugar portion of novelnucleoside analogs is provided. This synthesis can be used to make bothpurine and pyrimidine derivatives. The key step in the synthetic routeis the fluorination of a chiral, non-carbohydrate sugar ring precursor(4S)-5-(protected-oxy)-pentan-4-olide, for example,(4S)-5-(t-butyldiphenylsiloxy)-pentan-4-olide 4 using an electrophilicfluorine source, including, but not limited to,N-fluoro-(bis)benzenesulfonimide 5. This relatively new class ofN-fluorosulfonimide reagents was originally developed by Barnette in1984 and since then has seen much refinement and use as a convenient andhighly reactive source of electrophilic fluorine (Barnette, W. E. J. Am.Chem. Soc. 1984, 106, 452; Davis, F. A.; Han; W., Murphy, C. K. J. Org.Chem. 1995, 60, 4730; Snieckus, V.; Beaulieu, F.; Mohri, K.; Han, W.;Murphy, C. K.; Davis, F. A. Tetrahedron Lett. 1994, 35(21), 3465). Mostoften, these reagents are used to deliver fluorine to nucleophiles suchas enolates and metalated aromatics (Davis, F. A.; Han; W., Murphy, C.K. J. Org. Chem. 1995, 60, 4730). Specifically,N-fluoro-(bis)benzenesulfonimide (NFSi) is an air stable, easily handledsolid with sufficient steric bulk to stereoselectively fluorinate theetiolate of silyl-protected lactone 4. As a nonlimiting example of thisprocess, the synthesis of fluorolactone 6 and its use as a commonintermediate in the synthesis of a number of novel α-2′-fluoronucleosides is described in detail below. Given this description, one ofordinary skill can routinely modify the process as desired to accomplisha desired objective and to prepare a compound of interest.

Any source of electrophilic fluorine can be used that fluorinates theprecursor (4S)-5-(protected-oxy)-pentan-4-olide, for example,(4S)-5-(t-butyl-diphenylsiloxy)pentan-4-olide. Alternative sources ofelectrophilic fluorine include N-fluorosulfams (Differding, et al, Tet.Lett. Vol. 29, No. 47 pp 6087-6090 (1988); Chemical Reviews, 1992, Vol92, No. 4 (517)), N-fluoro-O-benzenedisulfonimide (Tet. Lett. Vol. 35,pages 3456-3468 (1994), Tet. Lett. Vol 35. No. 20, pages 3263-3266(1994)); J. Org. Chem. 1995, 60, 4730-4737), 1-fluoroethene andsynthetic equivalents (Matthews, Tet. Lett. Vol. 35, No. 7, pages1027-1030 (1994); Accufluor fluorinating agents sold by Allied Signal,Inc., Buffalo Research Laboratory, Buffalo, N.Y. (NFTh(1-fluoro-4-hydroxy-1,4-diazoa-bicyclo[2.2.2]octanebis(tetrafluoroborate)), NFPy (N-fluoropyridinium pyridineheptafluorodiborate), and NFSi (N-fluorobenzenesulfonimide);electrophilic fluorinating reagents sold by Aldrich Chemical Company,Inc., including N-fluoropyridinium salts((1-fluoro-2,4,6-trimethylpyridinium triflate,3,5-dichloro-1-fluoropyridinium triflate, 1-fluoropyridinium triflate,1-fluoropyridinium tetrafluoroborate, and 1-fluoropyridinium pyridineheptafluorodiborate) see also J. Am. Chem. Soc., Vol 112, No. 23 1990);N-fluorosulfonimides and -amides(N-fluoro-N-methyl-p-toluenesulfonamide,N-fluoro-N-propyl-p-toluenesulfonamide, and N-fluorobenzenesulfonimide);N-fluoro-quinuclidinium fluoride (J. Chem. Soc. Perkin Trans I 1988,2805-2811); perfluoro-2,3,4,5-tetrahydropyridine andperfluoro-(1-methylpyrrolidine), Banks, Cheng, and Haszeldine,Heterocyclic Polyfluoro-Compounds Part II (1964); 1-fluoro-2-pyridone,J. Org. Chem., 1983 48, 761-762; quaternary stereogenic centerspossessing a fluorine atom (J. Chem. Soc. Perkin Trans. pages 221-227(1992)); N-fluoro-2,4,6-pyridinium triflate, Shimizu, Tetrahedron Vol50(2), pages 487-495 (1994); N-fluoropyridinium pyridineheptafluorodiborate, J. Org. Chem. 1991, 56, 5962-5964; Umemoto, et al.,Bull. Chem. Soc. Jpn., 64 1081-1092 (1991);N-fluoroperfluoroalkylsulfonimides, J. Am. Chem. Soc., 1987, 109,7194-7196; Purrington, et al., Lewis Acid Mediated Fluorinations ofAromatic Substrates, J. Org. Chem. 1991, 56, 142-145.

A significant advantage of this methodology is the ability to accessseparately either the “natural” (1a) D or the “unnatural” (1b) Lenantiomer of the nucleosides by appropriate choice of L- or D-glutamicacid starting material, respectively.

Lactone 4 was synthesized by the route shown in Scheme 1 from L-glutamicacid as described by Ravid et al. (Tetrahedron 1978, 34, 1449) andTaniguchi et al. (Tetrahedron 1974, 30, 3547).

The enolate of lactone 4, prepared at −78° C. with LiHMDS in THF, isknown to be stable. Several syntheses using this enolate have beenperformed, including addition of electrophiles such asdiphenyldiselenide, diphenyldisulfide, and alkyl halides in high yield(Liotta, D. C.; Wilson, L. J. Tetrahedron Lett. 1990, 31(13), 1815; Chu,C. K.; Babu, J. R.; Beach, J. W.; Ahn, S. K.; Huang, H.; Jeong, L. S.;Lee, S. J. J. Org. Chem., 1990, 55, 1418; Kawakami, H.; Ebata, T.;Koseki, K.; Matsushita, H.; Naoi, Y.; Itoh, K. Chem. Lett. 1990, 1459).However, addition of a THF solution of 5 to the enolate of 4 gave pooryields of the desired monofluorinated product 6. Numerous by-productswere formed including what was surmised to be a difluorinated lactonethat is inseparable from other impurities. For this reason, the order ofaddition of the reagents was altered such that lactone 4 and NFSi 5 weredissolved together in THF and cooled to −78° C. Slow addition of LiHMDSresulted in a reaction yielding 6 as the only product in addition to asmall amount of unreacted stalling material (eq 1).

Equation 1

Fluorolactone 6 could be obtained in 50-70% yield after silica gelcolumn chromatography and crystallization. This reaction yielded asingle diastereomer of 6, presumably due to the interaction of thesterically bulky TBDPS group and the bulky fluorinating reagent 5.Identification of fluorolactone 6 as the α or “down” fluoro isomer wasaccomplished by comparison to previously published NMR data and by X-raycrystal structure determination of its enantiomer 20.

Lactone 6 was transformed into the anomeric acetate 8 as shown in Scheme2. It is of interest to note that lactol 7 exists exclusively as the βanomer and that acetate 8 shows no detectable α anomer by NMR, asreported by Niihata et al. (Bull. Chem. Soc. Jpn. 1995, 68, 1509).

Coupling of 8 with silylated pyrimidine bases was performed by standardVorbruggen methodology (Tetrahedron Lett. 1978, 15, 1339) using TMStriflate as the Lewis acid. Alternatively, any other Lewis acid known tobe useful to condense a base with a carbohydrate to form a nucleosidecan be used, including tin chloride, titanium chloride, and other tin ortitanium compounds. A number of bases were successfully coupled in highyields ranging from 72%-100% after column chromatography (eq 2, Table1).

Equation 2

TABLE 1 Glycosylation of Substituted Pyrimidines with 8 Cmpd. R₁ R₂yield 9 F OH 87% 10 F NH₂ 99% 11 H NHAc 91% 12 H NH₂ 72% 13 CH₃ OH 89%

Proton NMR indicated that the ratio of β to α nucleoside anomers wasapproximately 2:1 in all cases. The silyl protected nucleosides couldnot be resolved by column chromatography into the separate anomers.However, after deprotection of the 5′-oxygen with NH₄F in methanol (eq3), the α and β anomers could be readily separated and fee results aresummarized in Table 2.

Equation 3

TABLE 2 Deprotection of Nucleosides R₁ R₂ a yield b yield F OH 14a 19%14b 48% F NH₂ 15a 27% 15b 51% H NHAc 16a 17% 16b 31% H NH₂ 17a — 17b —CH₃ OH 18a 12% 18b 33%

The classification of the free nucleosides as α or β was based on thechemical shift of the anomeric proton (Table 3) and on the polarity ofthe nucleosides as observed by thin layer chromatography. A trend forall of the α/β pairs of free nucleosides was observed in that the lesspolar compound of the two had an anomeric proton chemical shift that wasnotably upfield from that of the more polar compound.

TABLE 3 Anomeric Proton Chemical Shift (ppm) Cmpd. α β 14 a, b 6.11 5.8915 a, b 6.08 5.92 16 a, b 6.09 5.90 17 a, b 6.05 5.92 18 a, b 6.11 5.93The correlation between anomeric proton chemical shift and absolutestructure was verified by comparison of 18a (Niihata, S.; Ebata, T.;Kawakami, H.; Matsushida, H. Bull. Chem. Soc. Jpn. 1995, 68, 1509) and18b (Aerschot, A. V.; Herdewijn, P.; Balzarini, J.; Pauwels, R.; DeClereq, E. J. Med. Chem. 1989, 32, 1743) with previously publishedspectral data and through X-ray crystal structure determination of 14band 15b. This finding is the opposite of the usual trend for nucleosidesin which the α anomer is normally the less polar of the two. Presumably,in the “down” 2″-fluorinated nucleosides, the strong dipole of the C—Fbond opposes the C—N anomeric bond dipole in the β isomer and reducesthe overall molecular dipole. Conversely, the α anomer has a geometrythat allows reinforcement of the molecular dipole through the additionof the C—F and C—N bond dipoles. Thus, the α anomer is more polar thanthe β anomer in the case of α-2′-fluoro nucleosides.

The α and β anomers 17a and 17b could not be separated by columnchromatography because the free amino group caused the nucleosides tostreak on silica gel. Therefore, it was necessary to useN⁴-acetylcytosine to prepare 11 and then resolve 16a and 16b. TheN⁴-acetyl group was removed quantitatively with a saturated solution ofammonia in methanol in order to obtain separated 17a and 17b. When5-fluorocytosine was used as the base (compound 10), the anomers 15a and15b were easily separated and no streaking on silica gel was observed.

Of the ten nucleosides listed in Table 2, it appears that only 17b(Martin, J. A.; Bushnell, D. J.; Duncan, I. B.; Dunsdon, S. J.; Hall, M.J.; Machin, P. J.; Merrett, J. H.; Parkes, K. E. B.; Roberts, N. A.;Thomas, G. J.; Galpin, S. A.; Kinchington, D. J. Med. Chem. 1990, 33(8),2137; Zenchoff, G. B.; Sun, R.; Okabe, M. J. Org. Chem. 1991, 56, 4392),18a (Niihata, S.; Ebata, T.; Kawakami, H.; Matsushida, H. Bull. Chem.Soc. Jpn. 1995, 68, 1509), and 18b (Aerschot, A. V.; Herdewijn, P.;Balzarini, J.; Pauwels, R.; De Clercq, E. J. Med. Chem. 1989, 32, 1743)have been synthesized previously. They, like the numerous known 2′-β or“up” fluoro nucleoside analogs¹⁴ have been synthesized from naturalprecursors (i.e., they are in the β-D configuration). It appears that noβ-L-2′-fluoro-ribofuranosyl nucleosides have been identified in theliterature prior to this invention.

Fluorine is usually introduced into these molecules through nucleophilicattack on an anhydro-nucleoside (Mengel, R.; Guschlbauer, W. Angew.Chem., Int. Ed. Engl. 1978, 17, 525) or through replacement andinversion of a stereochemically fixed hydroxyl group withdiethylaminosulfur trifluoride (DAST) (Herdewijn, P.; Aerschot, A. V.;Kerremans, L. Nucleosides Nucleotides 1989, 8(1), 65). One advantage ofthe present methodology is that no hydroxyl group is needed for fluorineintroduction. Thus, the process is not limited to natural nucleosides orsugars as starting materials, and provides an easy to access theunnatural enantiomers of the 2″-fluoro nucleosides.

Accordingly, several unnatural nucleosides were synthesized using thissynthetic route with D-glutamic acid 19 as the starting material (Scheme3). The sugar ring precursor 20 was fluorinated in the manner describedabove and coupled with various silylated bases (Table 4).

TABLE 4 Yields of Unnatural Nucleoside Analogs yield cmpd. (23-25) R₁ R₂a yield b yield 23 87% CH₃ OH 26a 24% 26b 61% 24 85% F OH 27a 35% 27b51% 25 99% F NH₂ 28a 34% 28b 52%

Successful synthesis of 29, as shown in Scheme 4, allows access to twocategories of nucleosides. The first is the class of compounds known as2′,3′-dideoxy-2′,3′-didehydro-2-2′-fluoro-nucleosides, 30, and thesecond is the “up”-fluoro or arabino analogs, 31, of the nucleosidesdescribed in Scheme 5 below.

Compounds 30 and 31 may be synthesized from a common intermediate 32,which may be accessed through selenylation of fluoroglycal 29.

Selenylated compound 32 may be transformed into the “up” fluoro analog31 through reduction with Raney nickel. Alternatively, oxidation of theselenide 32 with NaIO₄ or hydrogen peroxide followed by thermalelimination of the selenoxide intermediate lead to 30. Both of thesetransformations on the unfluorinated systems are well documented andhave been reported (Wurster, J. A.; Ph.D. Thesis, Emory University,1995; Wilson, L. J.; Ph.D. Thesis, Emory University, 1992).

In addition, the synthesis of the enantiomers of nucleosides 30 and 31is also possible since they arise from the enantiomer of 29.

An alternative route for the preparation of compounds of the typerepresented by 30, the2′,3′-dideoxy-2′,3′-didehydro-2′-fluoro-nucleosides, is shown in Scheme7. This route provides simple, direct access to this class of compoundsutilizing a wide range of silylated bases and has been successfullycompleted.

Formation of silyl ketene acetal from 6 allows for the stereoselectiveaddition of phenyl selenium bromide to generate compound 36 as a singleisomer. Reduction and acetylation of this compound proceeds smoothly andin high yield over the two steps to 37. The α orientation of the phenylselenyl group allows for stereoselection in the subsequent glycosylationstep, and synthesis of the β isomer of the nucleoside 38 is accomplishedin good yield. Compound 38 may be oxidized with hydrogen peroxide indichloromethane to yield the elimination product 39, but in ourexperience, it was merely necessary to adsorb 38 onto silica gel andallow to stand for several hours, after which time 39 could be elutedfrom a plug column in nearly quantitative yield. Removal of theprotected group from 39 to obtain the final compound 30 was performed asbefore and resulted in a good yield (81%) of product nucleoside.

The same series of chemical transformations that were used for thesynthesis of 30 and 31 may also be used for the synthesis of 34 and 35.

Experimental Section

General Procedures:

N-Fluoro-(bis)benzenesulfonimide 5 was obtained from Allied Signal, andwas used without further purification. All other reagents were obtainedfrom Aldrich Chemical Company and were used without furtherpurification. Melting points were determined on a Thomas Hoovercapillary melting point apparatus and are uncorrected. IR spectra wereobtained on a Nicolet Impact 400 FT-IR spectrometer. ¹H NMR and ¹³C NMRspectra were recorded on either NT-360 or Varian 400 MHz spectrometer.TLC plates were silica gel 60 F₂₅₄ (0.25 mm thickness) purchased from EMScience. Flash chromatography was carried out with silica gel 60(230-400 mesh ASTM) from EM Science. All reactions were performed inflame-dried glassware under an atmosphere of dry argon. Solvents wereremoved by rotary evaporation. Elemental analyses were performed byAtlantic Microlab, Inc, Atlanta, Ga.

(2S,4R)-5-(t-butyldiphenylsiloxy)-2-fluoropentan-4-olide (20). To aflask was added (4R)-5-(t-butyldiphenylsiloxy)-pentan-4-olide (20.0 g,0.0564 mol, 1.0 eq.) and N-fluoro-(bis)benzenesulfonimide (NFSi) 5(17.80 g, 0.0564 mol, 1.0 eq.) in 250 mL of anhydrous THF. The solutionwas cooled to −78° C. and 68.0 mL (0.0680 mol, 1.2 eq.) of a 1.0 Msolution of LiHMDS in THF was added dropwise over a period of 1 hr. Thiswas allowed to stir at −78° C. for an additional 2 hrs. and was thenwarmed to room temperature to stir for one hour. After completion, thereaction was quenched with 10 mL of saturated NH₄Cl solution. Themixture was diluted with three volumes of diethyl ether and was pouredonto an equal volume of saturated NaHCO₃. The organic layer was washed asecond time with saturated NaHCO₃ and once with saturated NaCl. Theorganic layer was dried over MgSO₄, filtered, and concentrated to alight yellow oil. The oil was purified by silica gel columnchromatograpy using a 30% diethyl ether/70% hexanes solvent system. Theresultant white solid was then crystallized from hot hexanes to yield13.04 g (62% yield) of a transparent crystalline solid: R_(f) (30%diethyl ether/70% hexanes)=0.26; mp 115-116° C. ¹H NMR (360 MHz, CDCl₃)d 7.63-7.60 (m, 4H), 7.45-7.35 (m, 6H), 5.49 (dt, J=52.9 and 7.9 Hz,1H), 4.69 (d, J=9.36 Hz, 1H), 3.91 (d, J=11.5 Hz, 1H), 3.60 (d, J=11.5Hz, 1H), 2.72-2.40 (m, 2H), 1.05 (s, 9H); ¹³C NMR (100 MHz, CDCl₃) d172.1 (d, J=20.5 Hz), 135.5, 135.4, 132.3, 131.7, 130.1, 128.0, 127.9,85.6 (d, J=186.6 Hz), 77.3 (d, J=5.3 Hz), 65.0, 31.8 (d, J=20.5 Hz),26.7, 19.1; IR (thin film) 2958, 1796, 1252, 1192, 1111, 1016 cm⁻¹; HRMScalculated for [M+Li] C₂₁H₂₅O₃FSiLi: 379.1717. Found: 379.1713. Anal.Calc. CHAFFS: C, 67.71; H, 6.76. Found: C, 67.72; H, 6.78.

5-O-(t-butyldiphenylsilyl)-2,3-dideoxy-2-fluoro-(L)-erythron-pentofuranose(21). To a flask was added lactone 20 (12.12 g, 0.0325 mol, 1.0 eq.) and240 mL of anhydrous THF. The solution was cooled to −78° C. and 65 mL(0.065 mol. 2.0 eq.) of a 1.0 M solution of DIBALH in hexanes was addeddropwise over a period of 30 min. This was allowed to stir at −78° C.for 3 hrs., after which time the reaction was quenched by the slowaddition of 2.93 mL (0.163 mol, 5.0 eq.) of water. The reaction wasallowed to warm to room temperature and stir for 1 hr., after which timea clear gelatinous solid formed throughout the entire flask. Thereaction mixture was diluted with two volumes of diethyl ether and waspoured onto an equal volume of saturated aqueous sodium potassiumtartrate solution in an Erlenmeyer flask. This was stirred for 20 min.until the emulsion had broken. The organic layer was separated and theaqueous layer was extracted three times with 250 mL of diethyl ether.The combined organic layers were dried over MgSO₄, filtered, andconcentrated to a light yellow oil. The product was purified by silicagel column chromatography using a 6:1 hexanes/ethyl acetate solventsystem. The resulting clear oil was crystallized from boiling hexanes togive 11.98 g (98% yield) of a white crystalline solid: R_(f) (30%diethyl ether/70% hexanes)=0.33; mp 66-67° C. ¹H NMR (360 MHz, CDCl₃) d7.68-7.66 (m, 4H), 7.55-7.38 (m, 6H), 5.39 (t, J=7.6 Hz, 1H), 4.99 (dd,J=52.2 and 4.3 Hz, 1H), 4.52 (m, 1H), 3.88 (dd, J=10.8 and 2.5 Hz, 1H),3.65 (d, J=7.9 Hz, 1H), 3.49 (dd, J=7.9 and 1.8 Hz, 1H), 2.44-2.07 (m,2H), 1.07 (s, 9H); ¹³C NMR (100 MHz, CDCl₃) d 135.7, 135.5, 132.2,132.1, 130.2, 130.0, 129.8, 127.9, 127.7, 99.8 (d, J=31.1 Hz), 96.6 (d,J=178.3 Hz), 79.4, 64.8, 29.9 (d, J=21.2 Hz), 26.8, 19.2; IR (thin film)3423, 2932, 1474, 1362, 1113 cm⁻¹; HRMS calculated for [M+Li]C₂₁H₂₇O₃FSiLi: 381.1874. Found: 381.1877. Anal. Calc. C₂₁H₂₇O₃FSi: C,67.35; H, 7.27. Found: C, 67.42; H, 7.31.

1-O-Acetyl -5-O-(t-butyldiphenylsilyl)-2,3-dideoxy-2-fluoro-(L)-erythron-pentofuranose(22). To a flask was added lactol 21 (8.50 g, 0.0227 mol, 1.0 eq.) and170 mL of anhydrous CH₂Cl₂. Then, DMAP (0.277 g, 0.00277 mol, 0.1 eq.)and acetic anhydride (13.5 mL, 0.143 mol, 6.3 eq.) were added andstirred at room temperature overnight. Upon completion, the reaction waspoured onto saturated NaHCO₃ solution. The organic layer was separated,and the aqueous layer was extracted three times with chloroform. Thecombined organic layers were dried over MgSO₄, filtered, and the solventremoved to yield a light yellow oil. The oil was purified by silica gelcolumn chromatography using an 8:1 hexanes/ethyl acetate solvent systemto give 9.85 g (99% yield) of a clear colorless oil: R_(f) (30% diethylether/70% hexanes)=0.44; ¹H NMR (360 MHz, CDCl₃) d 7.69-7.67 (m, 4H),7.43-7.38 (m, 6H), 6.30 (d, J=10.4 Hz, 1H), 5.06 (d, J=54.9 Hz, 1H),4.53 (m, 1H), 3.81 (dd, J=10.8 and 4.3 Hz, 1H), 3.72 (dd, J=10.8 and 4.3Hz, 1H), 2.38-2.12 (m, 2H), 1.89 (s, 3H), 1.07 (s, 9H); ¹³C NMR (100MHz, CDCl₃) d 169.4, 135.6, 135.5, 133.2, 133.1, 129.8, 129.7, 127.8,127.7, 99.3 (d, J=34.1 Hz), 95.5 (d, J=178.2 Hz), 81.4, 65.3, 31.6 (d,J=20.5 Hz), 26.8, 21.1, 19.3; IR (thin film) 3074, 2860, 1750, 1589,1229, 1113 cm⁻¹; HRMS calculated for [M−OCOCH₃] C₂₁H₂₆O₂FSi: 357.1686.Found: 357.1695. Anal. Calc. C₂₃H₂₉O₄FSi: C, 66.32; H, 7.02. Found: C,66.30; H, 7.04.

Representative procedure for the coupling of a silylated base with 22:(L)-5′-O-(t-butyldiphenylsilyl)-2′,3-dideoxy-2′-fluoro-5-fluorocytidine(25). To a flask equipped with a short-path distillation head was added5-fluorocytosine (2.01 g, 15.6 mmol, 5.0 eq), 35 mL of1,1,1,3,3,3-hexamethyldisilazane, and a catalytic amount (˜1 mg) of(NH₄)₂SO₄. The white suspension was heated to boiling for 1 hr. untilthe base was silylated and reaction was a clear solution. The excessHMDS was distilled off and the oily residue that remained was placedunder vacuum for 1 hr. to remove the last traces of HMDS. A white solidresulted which was dissolved, under argon, in 5 mL of anhydrous1,2-dichloroethane. To this clear solution was added a solution ofacetate 22 (1.30 g, 3.12 mmol, 1.0 eq.) in 5 mL of anhydrous1,2-dichloroethane. To this was added, at room temperature,trimethylsilyl trifluoromethanesulfonate (3.32 mL, 17.2 mmol, 5.5 eq.).The reaction was monitored by TLC (10% methanol/90% CH₂Cl₂) and wasobserved to be complete in 4 hrs. The reaction mixture was poured ontosaturated NaHCO₃. The organic layer was then separated, and the aqueouslayer was extracted three times with chloroform. The combined organiclayers were dried over MgSO₄, filtered, and the solvent removed to yielda white foam. The compound was purified by silica gel columnchromatography using a gradient solvent system from 100% CH₂Cl₂ to 10%methanol in CH₂Cl₂. The compound was isolated as 1.51 g (99% yield) of awhite foam: mixture of anomers R_(f) (100% EtOAc)=0.36; mp 74-80° C. ¹HNMR (400 MHz, CDCl₃) d 8.84 (bs, 1H), 8.04 (d, J=6.4 Hz, 0.67H),7.67-7.63 (m, 4H), 7.51-7.39 (m, 6.33H), 6.11 (d, J=20 Hz, 0.33H), 5.98(d, J=16.4 Hz, 0.67H), 5.88 (bs, 1H), 5.41 (d, J=52.4 Hz, 0.33H), 5.23(dd, J=50.4 and 4 Hz, 0.67H), 4.56 (m, 0.33H), 4.45 (m, 0.67H), 4.23(dd, J=12.0 and 1.6 Hz, 0.67H), 3.89 (dd, J=11.2 and 3.2 Hz, 0.33H),3.74-3.66 (m, 1H), 2.45-1.96 (m, 2H), 1.09 (s, 6H), 1.06 (s, 3H); ¹³CNMR (100 MHz, CDCl₃) d 158.6 (d, J=14.4 Hz), 158.4 (d, J=14.4 Hz),153.9, 153.8, 136.6 (d, J=240.5 Hz), 136.3 (d, J=239.7 Hz), 135.6,135.56, 135.5, 135.4, 133.1, 132.9, 132.5, 132.4, 130.1, 130.0, 129.9,127.9, 127.8, 125.8 (d, J=33.4 Hz), 124.6 (d, J=32.6 Hz), 96.5 (d,J=182.0 Hz), 91.7 (d, J=185.1), 90.7 (d, J=35.6 Hz), 87.7 (d, J=15.2Hz), 81.5, 79.5, 64.9, 63.0, 33.5 (d, J=20.5 Hz), 30.6 (d, J=20.4 Hz),26.9, 26.8, 19.22, 19.18; IR (thin film) 3300, 2960, 1682, 1608, 1513,1109 cm⁻¹; HRMS calculated for [M+Li]C₂₅H₂₉N₃O₃SiF₂Li: 492.2106. Found:492.2085. Anal. Calc. C₂₅H₂₉N₃O₃SiF₂·½ H₂O: C, 60.71; H, 6.11; N, 8.50.Found: C, 60.67; H, 6.03; N, 8.44.

Representative Procedure for the deprotection of silyl-protectednucleosides: α- and β-(L)-2′, 3′-dideoxy-2′-fluoro -5-fluoro cytidine(28a and 28b): Nucleoside 25 (1.098 g, 2.26 mmol, 1.0 eq.) was dissolvedin 15 mL of methanol to which was added ammonium fluoride (0.838 g, 22.6mmol, 10.0 eq.). This was stirred vigorously for 24 hrs., after whichtime TLC (15% ethanol/85% ethyl acetate) revealed that the reaction wascomplete. The reaction mixture was diluted with three volumes of ethylacetate and was filtered through a small (1 cm) silica gel plug. Theplug was rinsed with 200 mL of 15% ethanol/85% ethyl acetate solutionand the solvent was removed to yield a white foam. The compound waspurified by silica gel column chromatography using a 15% ethanol/85%ethyl acetate solvent system which also effected the separation of the αand β anomers. The yield of a as a white foam was 0.190 g (0.768 mmol,34% yield) and the yield of β as a white foam was 0.290 g (1.17 mmol,52% yield): (28a) R_(f) (15% EtOH , 85% EtOAc)=0.22; mp 199-203° C.(dec.). ¹H NMR (400 MHz, CD₃OD) d 7.78 (d, J=6.8 Hz, 1H), 6.07 (d,J=19.2 Hz, 1H), 5.37 (d, J=54.0 Hz, 1H), 4.60 (m, 1H), 3.80 (dd, J=12.0and 3.2 Hz, 1H), 3.56 (dd, J=12.4 and 4.4 Hz, 1H), 2.40-2.00 (m, 2H);¹³C NMR (100 MHz, DMSO-d₆) d 157.7 (d, J=13.6 Hz), 153.2, 135.9 (d,J=239.0 Hz), 126.2 (d, J=31.1 Hz), 92.4 (d, J=183.6 Hz), 86.7 (d, J=15.2Hz), 79.6, 62.7, 33.3 (d, J=20.5 Hz); IR (KBr) 3343, 3100, 1683, 1517,1104 cm⁻¹; HRMS calculated for [M+Li] C₉H₁₁N₃O₃F₂Li: 254.0929. Found:254.0919. Anal. Calc. C₉H₁₁N₃O₃F₂·½ H₂O: C, 42.19; H, 4.72; N, 16.40.Found: C, 42.44; H, 4.56: N, 16.56. (28b) R_(f) (15% EtOH, 85%EtOAc)=0.37; mp 182-186° C. (dec). ¹H NMR (400 MHz, DMSO-d₆) d 8.32 (d,J=7.6 Hz, 1H), 7.79 (bs, 1H), 7.53 (bs, 1H), 5.81 (d, J=16.8 Hz, 1H),5.37 (t, J=4.8 Hz), 5.18 (dd, J=51.6 and 3.2 Hz, 1H), 4.32 (m, 1H), 3.88(dd, J=12.0 and 2.8 Hz, 1H), 3.59 (dd, J=12.4 and 2.4 Hz, 1H), 2.20-1.99(m, 2H); ¹³C NMR (100 MHz, DMSO-d₆) d 157.7 (d, J=13.7 Hz), 153.2, 136.1(d, J=237.4 Hz), 125.3 (d, J=33.4 Hz), 97.3 (d, J=176.8 Hz), 89.9 (d,J=35.7 Hz), 81.6, 60.2, 30.3 (d, J=19.7 Hz); IR (KBr) 3487, 2948, 1678,1509, 1122 cm⁻¹; HRMS calculated for [M+Li] C₉H₁₁N₃O₃F₂Li: 254.0929.Found: 254.0935. Anal. Calc. C₉H₁₁N₃O₃F₂: C, 43.73; H, 4.49; N, 17.00.Found: C, 43.69; H, 4.53; N, 16.92.

(D)-5′-O-(t-butyldiphenylsilyl)-2′,3′-dideoxy-2′-fluoro-5-fluorouridine(9). mixture of anomers R_(f) (1:1 hexanes/EtOAc)=0.48; mp 65-70° C. ¹HNMR (400 MHz, CDCl₃) d 10.0 (bm, 1H), 7.99 (d, J=5.6 Hz, 0.63H), 7.65(m, 4H), 7.42 (m, 6.37H), 6.12 (dd, J=18.0 and 1.6 Hz, 0.37H), 6.00 (d,J=16 Hz, 0.63H), 5.37 (dd, J=54.6 and 2.4 Hz, 0.37H), 5.22 (dd, J=50.4and 4 Hz, 0.63H), 4.57 (m, 0.37H), 4.44 (m, 0.63H), 4.22 (dd, J=12.2 and2.0 Hz, 0.63H), 3.92 (dd, J=11.2 and 3.2 Hz, 0.37H), 3.70 (m, 1H), 2.22(m, 2H), 1.09 (s, 5.67H), 1.074 (s, 3.33H); ¹³C NMR (100 MHz, CDCl₃) d157.2 (d, J=31.7 Hz), 157.1 (d, J=25.8 Hz), 149.1, 148.8, 140.4 (d,J=236.6 Hz), 140.1 (d, J=235.2 Hz), 135.6, 135.5, 135.4, 132.9, 132.7,132.4, 132.3, 130.1, 130.0, 129.9, 127.9, 127.8, 125.1 (d, J=34.9 Hz),123.6 (d, J=34.1 Hz), 96.4 (d, J=182.0 Hz), 92.0 (d, J=185.9 Hz), 90.2(d, J=37.2 Hz), 87.0 (d, J=15.2 Hz), 81.7, 79.8, 64.8, 63.0, 33.3 (d,J=21.2 Hz), 31.0 (d, J=21.2 Hz), 26.9, 26.8, 19.2; IR (thin film) 3185,1722, 1117 cm⁻¹; HRMS calculated for [M+1] C₂₅H₂₉N₂O₄SiF₂: 487.1866.Found: 487.1853. Anal. Calc. C₂₅H₂₈N₂O₄SiF₂: C, 61.71; H, 5.80; N, 5.76.Found: C, 61.72; H, 5.86; N, 5.72.

(D)-5′-O-(t-butyldiphenylsilyl)-2′,3′-dideoxy-2′-fluoro-5-fluorocytidine(10) mixture of anomers R_(f) (100% EtOAc)=0.36; mp 75-81° C. ¹H NMR(400 MHz, CDCl₃) d 8.50 (bm, 1H), 8.05 (d, J=6.0 Hz, 0.67H), 7.67-7.63(m, 4H), 7.51-739 (m, 6.33H), 6.10 (d, J=20 Hz, 0.33H), 5.98 (d, J=16.4Hz, 0.67H), 5.62 (bm, 1H), 5.41 (d, J=52.4 Hz, 0.33H), 5.23 (dd, J=51.6and 4 Hz, 0.67H), 4.57 (m, 0.33H), 4.48 (m, 0.67H), 4.24 (dd, J=12.4 and2.0 Hz, 0.67H), 3.89 (dd, J=11.2 and 3.2 Hz, 0.33H), 3.74-3.66 (m, 1H),2.39-1.95 (m, 2H), 1.09 (s, 6H), 1.06 (s, 3H); ¹³C NMR (100 MHz, CDCl₃)d 158.4 (d, J=14.4 Hz), 158.3 (d, J=15.2 Hz), 153.8, 153.7, 136.5 (d,J=240.5 Hz), 136.2 (d, J=241.8 Hz), 135.59, 135.56, 135.4, 133.0, 132.9,132.5, 132.4, 130.1, 130.0, 129.9, 127.9, 127.8, 124.8 (d, J=31.9 Hz),96.5 (d, J=181.3 Hz), 91.8 (d, J=175.2 Hz), 90.7 (d, J=24.9 Hz), 87.8(d, J=21.2 Hz), 81.6, 79.6, 64.9, 63.0, 33.5 (d, J=19.7 Hz), 30.6 (d,J=21.3 Hz), 26.9, 26.8, 19.2, 14.2; IR (thin film) 3304, 2959, 1680,1621, 1508, 1105 cm⁻¹; HRMS calculated for [M+Li] C₂₅H₂₉N₃O₃SiF₂Li:492.2106. Found: 492.2110. Anal. Calc. C₂₅H₂₉O₃SiF₂: C, 61.84; H, 6.02;N, 8.65. Found: C, 61.86; H, 6.09; N, 8.55.

(D)-N⁴-acetyl-5′-O-(t-butyldiphenylsilyl)-2′,3′-dideoxy-2′-fluoro-cytidine(11). mixture of anomers R_(f) (15% EtOH , 85% EtOAc)=0.75; mp 81-86° C.¹H NMR (400 MHz, CDCl₃) d 10.58 (bs, 1H), 8.40 (d, J=7.2 Hz, 0.61H),7.86 (d, J=7.6 Hz, 0.38H), 7.67-7.65 (m, 4H), 7.51-7.41 (m, 6H), 7.27(d, J=8.4 Hz, 1H), 6.12 (t, J=15.8 Hz, 1H), 5.51 (d, J=52.6 Hz, 0.38H),5.21 (dd, J=50.8 and 2.9 Hz, 0.61H), 4.62 (m, 0.38H), 4.54 (m, 0.61H),4.28 (d, J=11.5 Hz, 0.61H), 3.95 (dd, J=11.9 and 3.2 Hz, 0.38H),3.79-3.70 (m, 1H), 2.46-2.04 (m, 5H), 1.12 (s, 5.49H), 1.07 (s, 3.42H);¹³C NMR (100 MHz, CDCl₃) d 171.5, 171.3, 163.4, 154.9, 144.9, 144.1,135.5, 135.4, 133.0, 132.8, 132.5, 132.2, 130.2, 130.1, 129.9, 128.0,127.8, 96.8 (d, J=91.1 Hz), 96.2 (d, J=147.9 Hz), 92.3, 91.2 (d, J=35.7Hz), 90.5, 88.5 (d, J=15.9 Hz), 81.9, 80.1, 64.7, 62.9, 33.5 (d, J=20.5Hz), 30.5 (d, J=20.5 Hz), 26.9, 26.8, 24.9, 24.8, 19.3, 19.2; IR (thinfilm) 3237, 2932, 1722, 1671, 1559, 1493, 1107 cm⁻¹; HRMS calculated for[M+Li]C₂₇H₃₂N₃O₄FSiLi: 516.2306. Found: 516.2310. Anal. Calc.C₂₇H₃₂N₃O₄FSi: C, 63.63; H, 6.33; N, 8.24. Found: C, 63.45; H, 6.42; N,8.09.

(D)-5′-O-(t-butyldiphenylsilyl)-2′,3′-dideoxy-2′-fluoro-cytidine (12).mixture of anomers R_(f) (15% EtOH , 85% EtOAc)=0.50; mp 98-104° C. ¹HNMR (360 MHz, CDCl₃) d 7.9 (d, J=7.2 Hz, 0.64H, H-6), 7.65 (m, 4H),7.47-7.38 (m, 6.36H), 6.15 (d, J=20.5 Hz, 0.36H), 6.05 (d, J=16.6 Hz,0.64H), 5.83 (d, J=7.9 Hz, 0.36H), 5.46 (d, J=7.2 Hz, 0.64H), 5.30-5.10(m, 1H), 4.55 (m, 0.36H), 4.44 (m, 0.64H), 4.22 (d, J=9.7 Hz, 0.64H),3.88-3.63 (m, 1.36H), 2.38-1.95 (m, 2H), 1.09 (s, 5.76H), 1.06 (s,3.24H); ¹³C NMR (100 MHz, CDCl₃) d 166.1, 155.8, 141.5, 140.5, 135.6,135.4, 133.1, 132.9, 132.8, 132.4, 130.1, 130.0, 129.8, 128.0, 127.9,127.8, 96.7 (d, J=181.3 Hz), 93.4 (d, J=140.3 Hz), 94.5, 90.8 (d, J=35.6Hz), 90.8, 87.8 (d, J=15.9 Hz), 81.2, 79.4, 65.0, 63.2, 33.7 (d, J=21.2Hz), 30.8 (d, J=20.4 Hz), 26.9, 26.8, 19.3, 19.2; IR (thin film) 3470,3339, 1644, 1487, 1113 cm⁻¹; HRMS calculated for [M+Li] C₂₅H₃₀N₃O₃FSiLi:474.2201. Found: 474.2198. Anal. Calc. C₂₅H₃₀N₃O₃FSi: C, 64.21; H, 6.47;N, 8.99. Found: C, 64.04; H, 6.58; N, 8.76.

α-(D)-2′,3′-Dideoxy-2′-fluoro-5-fluorouridine (14a). R_(f) (100%EtOAc)=0.38; mp 153-155° C. ¹H NMR (360 MHz, CD₃OD) d 7.80 (d, J=6.8 Hz,1H), 6.11 (d, J=18.7 Hz, 1H), 5.35 (d, J=52.9, 1H), 4.59 (m, 1H), 3.81(d, J=11.9 Hz, 1H), 3.57 (dd, J=12.6 and 3.6 Hz, 1H), 2.36-2.15 (m, 2H);¹³C NMR (100 MHz, CD₃OD) d 159.6 (d, J=25.8 Hz), 150.7, 141.5 (d,J=230.6 Hz), 127.0 (d, J=34.9 Hz), 93.9 (d, J=185.1 Hz), 88.5 (d, J=15.1Hz), 81.8, 64.3, 34.3 (d, J=20.5 Hz); IR (KBr) 3421, 3081, 1685, 1478,1111 cm⁻¹; HRMS calculated for [M+Li] C₉H₁₀N₂O₄F₂Li: 255.0769. Found:255.0778. Anal. Calc. C₉H₁₀N₂O₄F₂: C, 43.56; H, 4.06; N, 11.29. Found:C, 43.59; H, 4.11; N, 11.17.

β-(D)-2′,3′-Dideoxy-2′-fluoro-5-fluorouridine (14b). R_(f) (100%EtOAc)=0.54; mp 152-154 (C. ¹H NMR (360 MHz, CD₃OD) d 8.41 (d, J=7.2 Hz,1H), 5.89 (d, J=16.6 Hz, 1H), 5.21 (dd, J=51.5 and 3.6 Hz, 1H), 4.41 (m,1H), 4.00 (d, J=12.6 Hz, 1H), 3.67 (d, J=12.2 Hz, 1H), 2.25-2.09 (m,2H); ¹³C NMR (100 MHz, CD₃OD) d 159.7 (d, J=25.8 Hz), 150.7, 141.8 (d,J=229.8 Hz), 126.3 (d, J=36.4 Hz), 98.3 (d, J=179 Hz), 91.9 (d, J=37.1Hz), 83.6, 61.9, 31.9 (d, J=20.5 Hz); IR (KBr) 3417, 3056, 1684, 1474,1105 cm⁻¹; HRMS calculated for [M+Li]C₉H₁₀N₂O₄F₂Li: 255.0769. Found:255.0764. Anal. Calc. C₉H₁₀N₂O₄F₂: C, 43.56; H, 4.06; N, 11.29. Found:C, 43.37; H, 3.98; N, 11.22.

α-(D)-2′,3′-Dideoxy-2′-fluoro-5-fluorocytidine (15a). R_(f) (15% EtOH,85% EtOAc)=0.22; mp 198-202° C. (dec). ¹H NMR (400 MHz, CD₃OD) d 7.78(d, J=6.8 Hz, 1H), 6.07 (d, J=18.8 Hz, 1H), 5.37 (d, J=54.0 Hz, 1H),4.59 (m, 1H), 3.80 (dd, J=12.0 and 3.2 Hz, 1H), 3.57 (dd, J=12.4 and 4.4Hz, 1H), 2.38-2.14 (m, 2H); ¹³C NMR (100 MHz, CD₃OD) d 159.9 (d, J=13.6Hz), 156.5, 138.3 (d, J=240.4 Hz), 127.5 (d, J=33.4 Hz), 93.6 (d,J=184.3 Hz), 89.5 (d, J=15.9 Hz), 81.8, 64.4, 34.5 (d, J=20.5 Hz); IR(KBr) 3486, 3098, 1681, 1519, 1108 cm⁻¹; HRMS calculated for [M+Li]C₉H₁₁N₃O₃F₂Li: 254.0929. Found: 254.0929. Anal. Calc. C₉H₁₁N₃O₃F₂·½ H₂O:C, 42.19; H, 4.72; N, 16.40. Found: C, 41.86; H, 4.75; N, 16.36.

β-(D)-2′,3′-Dideoxy-2′-fluoro-5-fluorocytidine (15b). R_(f)(15% EtOH ,85% EtOAc)=0.37; mp 181-183° C. (dec). ¹H NMR (400 MHz, CD₃OD) d 8.45(d, J=7.2 Hz, 1H), 5.92 (dd, J=16.2 and 1.2 Hz, 1H), 5.18 (dd, J=50.8and 4.0 Hz, 1H), 4.46 (m, 1H), 4.05 (dd, J=12.4 and 2.4 Hz, 1H), 3.72(dd, J=12.8 and 2.4 Hz, 1H), 2.27-2.05 (m, 2H); ¹³C NMR (100 MHz, CD₃OD)d 159.9 (d, J=13.6 Hz), 156.5, 138.5 (d, J=240.5 Hz), 126.9 (d, J=33.4Hz), 98.4 (d, J=179.0 Hz), 92.5 (d, J=36.4 Hz), 83.6, 6.1.9, 31.6 (d,J=20.5 Hz); IR (KBr) 3494, 2944, 1689, 1522, 1106 cm⁻¹; HRMS calculatedfor [M+Li] C₉H₁₁N₃O₃F₂Li: 254.0929. Found: 254.0936. Anal. Calc.C₉H₁₁N₃O₃F₂: C, 43.73; H, 4.49; N, 17.00. Found: C, 43.84; H, 4.47; N,17.05.

α-(D)-N⁴-acetyl-2′,3′-dideoxy-2′-fluoro-cytidine (16a). R_(f) (15% EtOH,85% EtOAc)=0.40; mp 208-212° C. ¹H NMR (360 MHz, DMSO -d₆) d (10.91, bs,1H), 8.05 (d, J=7.2 Hz, 1H), 7.25 (d, J=7.2 Hz, 1H), 6.08 (dd, J=19.1and 2.9 Hz, 1H), 5.42 (d, J=52.2 Hz, 1H), 4.97 (bs, 1H), 4.54 (m, 1H),3.63 (d, J=13.0 Hz, 1H), 3.47 (d, J=13.3 Hz, 1H), 2.35-2.15 (m, 2H),2.11 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) d 171.0, 162.6, 154.3, 145.7,94.9, 92.0 (d, J=183.6 Hz), 87.5 (d, J=15.9 Hz), 80.2, 62.6, 33.3 (d,J=19.7 Hz), 24.4; IR (KBr) 3436, 3227, 1702, 1661, 1442, 1102 cm⁻¹; HRMScalculated for [M+Li] C₁₁H₁₄N₃O₄FLi: 278.1128. Found: 278.1136. Anal.Calc. C₁₁H₁₄N₃O₄F: C, 48.71; H, 5.20; N, 15.49. Found: C, 48.73; H,5.23; N, 15.52.

β-(D)-N⁴-acetyl-2′,3′-dideoxy-2′-fluoro-cytidine (16b). R_(f) (15% EtOH,85% EtOAc)=0.50; mp 174-178° C. ¹H NMR (360 MHz, DMSO-d₆) d (10.90, bs,1H), 8.46 (d, J=7.2 Hz, 1H), 7.18 (d, J=7.2 Hz, 1H), 5.90 (d, J=16.9 Hz,1H), 5.27 (d, J=52.9 Hz, 1H), 5.27 (bs, 1H), 4.39 (m, 1H), 3.88 (d,J=13.0 Hz, 1H), 3.61 (d, J=13.0 Hz, 1H), 2.09 (s, 3H), 2.20-1.85 (m,2H); ¹³C NMR (100 MHz, DMSO-d₆) d 171.0, 162.6, 154.4, 144.7, 97.0 (d,J=177.5 Hz), 95.0, 90.7 (d, J=36.6 Hz), 82.2, 60.3, 30.3 (d, J=19.7 Hz),24.3; IR (KBr) 3447, 3245, 1703, 1656, 1497, 1122 cm⁻¹; HRMS calculatedfor [M+Li] C₁₁H₁₄N₃O₄FLi: 278.1128. Found: 278.1133. Anal. Calc.C₁₁H₁₄N₃O₄F: C, 48.71: H, 5.20; N, 15.49. Found: C, 48.65; H, 5.22; N,15.46.

α-(D)-2′,3′-Dideoxy-2′-fluoro-cytidine (17a). R_(f) (15% EtOH, 85%EtOAc)=0.08; mp 234-237° C. (dec). ¹H NMR (400 MHz, DMSO-d₆) d 7.52 (d,J=7.6 Hz, 1H), 7.21 (bm, 2H), 6.05 (dd, J=20.4 and 3.2 Hz, 1H), 5.73 (d,J=7.2 Hz, 1H), 5.28 (d, J=52.4 Hz, 1H), 4.93 (t, J=5.6 Hz, 1H), 4.45 (m,1H), 3.58 (m, 1H), 3.43 (m, 1H), 2.26-2.13 (m, 2H); ¹³C NMR (100 MHz,DMSO-d₆) d 165.8, 155.0, 141.6, 93.3, 92.2 (d, J=182.8 Hz), 86.6 (d,J=15.1 Hz), 79.4, 62.8, 33.3 (d, J=19.7 Hz); IR (KBr) 3366, 3199, 1659,1399, 1122 cm⁻¹; HRMS calculated for [M+Li] C₉H₁₂N₃O₃FLi: 236.1023.Found: 236.1014. Anal. Calc. C₉H₁₂N₃O₃F: C, 47.16; H, 5.28; N, 18.33.Found: C, 47.40; H, 5.34; N, 18.51.

β-(D)-2′,3′-Dideoxy-2′-fluoro-cytidine (17b). Nucleoside 25 (0.160 g,0.59 mmol) was dissolved in 10 mL of saturated methanolic ammonia. Afterstirring for 5 min., the reaction was complete. The methanolic ammoniawas removed and the resultant white solid was placed under vacuum andheated gently in a 60° C. water bath for 2 hrs. to remove the acetamideby-product through sublimation. The white solid was crystallized from 5%methanol/95% methylene chloride to give a quantitative yield of a whitecrystalline solid. R_(f) (15% EtOH , 85% EtOAc)=0.18; mp 191-195° C.(dec). ¹H NMR (360 MHz, CD₃OD) d 8.10 (d, J=7.2 Hz, 1H), 5.92 (d, J=17.3Hz, 1H), 5.82 (d, J=7.6 Hz, 1H), 5.13 (d, J=50.0 Hz, 1H), 4.39 (m, 1H),3.97 (d, J=12.2 Hz, 1H), 3.68 (dd, J=13.0 and 2.5 Hz, 1H), 2.21-2.00 (m,2H); ¹³C NMR (100 MHz, CD₃OD) d 165.9, 155.0, 140.8, 97.3 (d, J=176.8Hz), 93.6, 90.3 (d, J=35.6 Hz), 81.3, 60.7, 31.0 (d, J=20.5 Hz); IR(KBr) 3397, 3112, 1680, 1400, 1178, 1070 cm⁻¹; HRMS calculated for[M+Li] C₉H₁₂N₃O₃FLi: 236.1024. Found: 236.1028. Anal. Calc. C₉H₁₂N₃O₃F:C, 47.16; H, 5.28; N, 18.33. Found: C, 47.01; H, 5.21; N, 18.29.

(L)-5′-O-(t-butyldiphenylsilyl)-2′,3′-dideoxy-2′-fluoro-thymidine (23).mixture of anomers R_(f) (10% MeOH/90% CH₂Cl₂)=0.56; mp 61-65° C. ¹H NMR(360, MHz, CDCl₃) d 9.48 (bs, 1H), 7.67 (m, 4H), 7.45-7.37 (m, 7H), 6.15(dd, J=20.2 and 3.2 Hz, 0.36H), 5.99 (d, J=18.4 Hz, 0.64H), 5.34 (d,J=51.8 Hz, 0.36H), 5.24 (dd, J=52.2 and 4.3 Hz, 0.64H), 4.59 (m, 0.36H),4.45 (m; 0.64H), 4.17 (dd, J=12.2 and 2.5 Hz, 0.64H), 3.91 (dd, J=11.9and 2.9 Hz, 0.36H), 3.81 (dd, J=11.5 and 2.9 Hz, 0.64H), 3.68 (dd,J=10.8 and 3.6 Hz, 0.36H), 2.40-2.12 (m, 2H), 1.94 (Ss 1.08H), 1.61 (s,1.92H), 1.10 (s, 5.76H), 1.07 (s, 3.24H); ¹³C NMR (100 MHz, CDCl₃)164.1, 164.0, 150.4, 150.2, 136.4, 135.6, 135.5, 135.4, 135.3, 135.2,133.0, 132.8, 132.6, 130.1, 130.0, 129.9, 127.94, 127.90, 127.8, 110.8,109.8, 96.4 (d, J=181.3 Hz), 92.1 (d, J=185.8 Hz), 90.7 (d, J=36.4 Hz),86.6 (d, J=15.2 Hz), 80.9, 79.4, 64.9, 63.6, 33.4 (d, J=20.5 Hz), 32.0(d, J=21.2 Hz), 27.0, 26.8, 19.4, 19.2, 12.6, 12.2; IR (thin film) 3183,3050, 1696, 1506, 1188 cm⁻¹; HRMS calculated for [M+Li] C₂₆H₃₁N₂O₄SiF:489.2197. Found: 489.2175. Anal. Calc. C₂₆H₃₁N₂O₄SiF: C, 64.71; H, 6.47;N, 5.80. Found: C, 64.88; H, 6.56; N, 5.76.

(L)-5′-O-(t-butyldiphenylsilyl)-2′,3′-dideoxy-2′-fluoro-5-fluorouridine(24). mixture of anomers R_(f) (1:1 hexanes/EtOAc)=0.48; mp 65-71° C. ¹HNMR (400 MHz, CDCl₃) d 9.08 (bs, 0.4H), 9.00 (bs, 0.6H) 8.01 (d, J=5.4Hz, 0.6H), 7.65 (m, 4H), 7.42 (m, 6.4H), 6.10 (dd, J=20.2 and 1.4 Hz,0.4H), 6.00 (d, J=16.0 Hz, 0.6H), 5.35 (dd, J=52.4 and 1.6 Hz, 0.4H),(5.22, dd, J=51.2 and 4 Hz, 0.6H), 4.57 (m, 0.4H), 4.44 (m, 0.6H), 4.22(dd, J=12.4 and 2.0 Hz, 0.6H), 3.91 (dd, J=11.2 and 2.9 Hz, 0.4H), 3.70(m, 1H), 2.45-2.00 (m, 2H), 1.09 (s, 5.4H), 1.07 (s, 3.6H); ¹³C NMR (100MHz, CDCl₃) d 156.9 (d, J=26.5 Hz), 148.8, 148.6, 140.3 (d, J=236.7 Hz),140.1 (d, J=235.1 Hz), 135.6, 135.5, 135.4, 132.9, 132.7, 132.4, 132.3,130.2, 130.1, 129.9, 127.9, 127.8, 125.1 (d, J=34.9 Hz), 123.6 (d,J=34.2 Hz), 96.4 (d, J=182.9 Hz), 92.0 (d, J=186.6 Hz), 90.2 (d, J=36.0Hz), 86.9 (d, J=15.1 Hz), 81.7, 79.8, 64.8, 63.0, 33.2 (d, J=20.5 Hz),30.9 (d, J=20.4 Hz), 26.9, 26.8, 19.2; IR (thin film) 3191, 1719, 1113cm⁻¹; HRMS calculated for [M+Li] C₂₅H₂₈N₂O₄SiF₂Li: 493.1946. Found:493.1952. Anal. Calc. C₂₅H₂₈N₂O₄SiF₂: C, 61.71; H, 5.80; N, 5.76. Found:C, 61.73; H, 5.83; N, 5.77.

α-(L)-2′,3′-Dideoxy-2′-fluoro-thymidine (26a). R_(f) (100% EtOAc)=0.25;mp 147-149° C. ¹H NMR (360 MHz, CD₃OD) d 7.45 (s, 1H), 6.11 (dd, J=19.4and 2.9 Hz, 1H), 5.30 (d, J=53.6 Hz, 1H), 4.58 (m, 1H), 3.79 (dd, J=12.2and 2.2 Hz, 1H), 3.55 (dd, J=12.2 and 3.6 Hz, 1H), 2.40-2.15 (m, 2H),1.87 (s, 3H); ¹³C NMR (100 MHz, CD₃OD) d 166.6, 152.3, 138.6, 110.5,93.9 (d, J=185.1 Hz), 88.3 (d, J=15.1 Hz), 81.7, 64.4, 34.5 (d, J=20.5Hz), 12.6; IR (KBr) 3436, 3166, 1727, 1667, 1362, 1186 cm⁻¹; HRMScalculated for [M+Li] C₁₀H₁₃N₂O₄FLi: 251.1019. Found: 251.1014. Anal.Calc. C₁₀H₁₃N₂O₄F: C, 49.18; H, 5.37; N, 11.47. Found: C, 49.32; H,5.40; N, 11.29.

β-(L)-2′,3′-dideoxy-2′-fluoro-thymidine (26b). R_(f) (100% EtOAc)=0.38;mp 186-188° C. ¹H NMR (360 MHz, CD₃OD) d 7.94 (s, 1H), 5.93 (d, J=17.6Hz, 1H), 5.20 (d, J=51.8 Hz, 1H), 4.40 (m, 1H), 3.98 (d, J=11.9 Hz, 1H),3.68 (d, J=13.0 Hz, 1H), 2.37-2.10 (m, 2H), 1.83 (s, 3H); ¹³C NMR (100MHz, CD₃OD) d 166.7, 152.3, 138.2, 111.0, 98.4 (d, J=178.3 Hz), 92.1 (d,J=36.4 Hz), 83.1, 62.4, 32.5 (d, J=20.5 Hz), 12.6; IR (KBr) 3478, 3052,1684, 1363, 1192, 1005 cm⁻¹: Anal. Calc. C₁₀H₁₃N₂O₄F: C, 49.18; H, 5.37;N, 11.47. Found: C, 49.29; H, 5.44; N, 11.36.

α-(L)-2′,3′-dideoxy-2′-fluoro-5-fluorouridine (27a). R_(f) (100%EtOAc)=0.38; mp 155-157° C.). ¹H NMR (400 MHz, CD₃OD) d 7.80 (d, J=6.8Hz, 1H), 6.13 (d, J=20.0 Hz, 1H), 5.35 (d, J=54.4 Hz, 1H), 4.63 (m, 1H),3.81 (dd, J=11.9 and 3.2 Hz, 1H), 3.58 (dd, J=12.4 and 2.0 Hz, 1H),2.41-2.15 (m, 2H); ¹³C NMR (100 MHz, CD₃OD) d 159.6 (d, J=25.8 Hz),150.7, 141.5 (d, J=230.6 Hz), 127.0 (d, J=34.9 Hz), 93.9 (d, J=184.3Hz), 88.5 (d, J=15.1 Hz), 81.9, 64.3, 34.3 (d, J=20.5 Hz); IR (KBr)3401, 3098, 1661, 1458, 1018 cm⁻¹; HRMS calculated for [M+Li]C₉H₁₀N₂O₄F₂Li: 255.0769. Found: 255.0771. Anal. Calc. C₉H₁₀N₂O₄F₂: C,43.56; H, 4.06; N, 11.29. Found: C, 43.70; H, 4.17; N, 11.15.

β-(L)-2′,3′-dideoxy-2′-fluoro-5-fluorouridine (27b). R_(f) (100%EtOAc)=0.54; mp 153-156° C. ¹H NMR (400 MHz, CD₃OD) d 8.46 (d, J=6.8 Hz,1H), 5.94 (d, J=16.4 Hz, 1H), 5.25 (dd, J=51.6 and 4.0 Hz, 1H), 4.41 (m,1H), 4.05 (dd, J=12.8 and 2.4 Hz, 1H), 3.72 (dd, J=12.4 and 2.4 Hz, 1H),2.34-2.09 (m, 2H); ¹³C NMR (100 MHz, CD₃OD) d 159.7 (d, J=25.8 Hz),150.7, 141.8 (d, J=230.6 Hz), 126.3 (d, J=35.7 Hz), 98.3 (d, J=184.6Hz), 91.9 (d, J=36.4 Hz), 83.6, 61.9, 31.9 (d, J=20.5 Hz); IR (KBr)3482, 3037, 1702, 1654, 1402, 1103 cm⁻¹; HRMS calculated for [M+Li]C₉H₁₀N₂O₄F₂Li: 255.0769. Found: 255.0764. Anal. Calc. C₉H₁₀N₂O₄F₂: C,43.56; H, 4.06; N, 11.29. Found: C, 43.59; H, 4.06; N, 11.17.

Preparation of L-2′-Fluoro-2′,3′-Unsaturated Nucleosides

A second facile synthesis of unsaturated 2′-fluoronucleosides has alsonow been accomplished and is described below. The synthesis involvesreacting a protected pyrimidine or purine base with key intermediate 309in the presence of a Lewis acid, as described generally in Scheme 9below. Representative compounds made according to this synthesis aredescribed in Tables 5-6.

TABLE 2 No. H-1′ H-3′ H-4′ H-5′ others 10^(a) 6.88 (m) 5.72 (m) 4.97(m), 4.88 (m) 3.80 (m) 8.02 (s, NH), 7.94 (d, H-6, J = 8 Hz), 7.18 (d,H-5, J = 8 Hz), 0.92 (s, ^(t)Bu), 0.90 (s, ^(t)Bu), 0.10, 0.09, 0.085,0.074 (4s, 4 × CH₃) 11^(a) 6.96 (s), 6.87 (m) 5.73 (s), 4.98 (m), 4.84(m) 3.76 (m) 8.15 (s, NH), 7.38 (s, H-6), 1.94 (s, 5-CH₃), 0.92 (s,^(t)Bu), 0.90 (s, ^(t)Bu), 5.66 (s) 0.10, 0.09, 0.085, 0.074 (4s, 4 ×CH₃) 12^(a) 7.12 (s) 5.61 (s) 4.94 (s) 3.88 (m) 8.41 (d, H-6, J = 7.2Hz), 7.71 (m, 6H, H-5, Ph-H), 0.94 (s, ^(t)Bu), 0.13, 0.12 (2s, 2 × CH₃)13^(a) 7.08 (ps t) 5.75 (s) 5.05 (ps t, J = 4.4, 3.76 (m) 7.91 (d, H-6,J = 6 Hz), 7.57 (m, 6H, H-5, Ph-H), 0.91 (s, ^(t)Bu), 0.09, 0.08, 4.8Hz) 0.07 (3s, 2 × CH₃) 14^(a) 15^(a) 16^(b) 6.77 (s) 6.01 (s) 4.81 (s)3.58 (s) 11.5 (s, —NH), 7.99 (d, H-6, J = 8 Hz), 5.71 (d, H-5, J = 8Hz), 5.13 (t, J = 5.2 Hz, OH) 17^(b) 6.77 (t, J = 4.4 Hz) 6.02 (d, 5.02(ps t, J = 4, 3.50 (m) 11.5 (s, —NH), 7.56 (d, H-6, J = 8 Hz), 5.70 (d,H-5, J = 8 Hz), J = 1.2 4.4 Hz) 4.94 (t, OH, J = 6 Hz) Hz) 18^(b) 6.77(s) 6.00 (s) 4.80 (s) 3.80 (s) 11.5 (s, —NH), 7.89 (s, H-6), 5.17 (t, J= 5.2 Hz, OH), 1.76 (s, 3H, CH₃-6) 19^(b) 6.78 (ps t, J = 4, 6.01 (s)5.05 (t, J = 4 Hz) 3.51 (m) 11.5 (s, —NH), 7.37 (s, H-5), 4.94 (t, J = 6Hz, OH), 1.81 (s, 3H, CH₃-6) 4.4 Hz) 20^(b) 7.01 (s) 5.71 (s) 4.99 (s)3.88 (m) 8.21 (d, J = 8 Hz, H-6), 7.71 (m, H-5, Ph-H) 21^(b) 7.16 (ps t,J = 3.6, 5.74 (s) 5.13 (ps t, J = 3.2, 3.79 (m) 7.92 (d, J = 7.2 Hz,H-6), 7.57 (m, H-5, Ph-H) 4.4 Hz) 4.8 Hz) 22^(b) 6.85 (s) 5.94 (d, 4.76(s) 3.56 (s) 7.86 (d, J = 7.2 Hz, H-6), 7.35, 7.32 (2s, NH₂), 5.77 (d, J= 7.2 Hz, H-5), J = 1.2 5.07 (t, J = 5.2 Hz, OH) Hz) 23^(b) 6.86 (ps t,J = 4.4, 5.94 (d, 4.94 (m) 3.49 (m) 7.47 (d, J = 7.6 Hz, H-6), 7.35,7.32 (2s, NH₂), 5.80 (d, J = 7.2 Hz, 4.8 Hz) J = 1.6 H-5) Hz))^(a)CDCl₃, ^(b)DMSO-d⁶

TABLE 3 No. H-1′ H-3′ H-4′ H-5′ Others 24^(a) 25^(a) 26^(a) 7.01 (s),6.93 (t, 5.85 (s), 5.78 (s) 5.18 (ps t, J = 4, 3.85 (m) 8.79, 8.78 (2s,H-8), 8.60, 8.21 (2s, H-2), 1.94 (s, 5-CH₃), 0.92, J = 4.4 Hz) 4.4 Hz),5.02 (s) 0.91 (2s, ^(t)Bu), 0.111, 0.105, 0.097, 0.095 (4s, 4 × CH₃)27^(a) 6.88 (s) 5.77 (s) 5.02 (s) 3.88 (m) 8.50 (s, H-8), 0.91 (s,^(t)Bu), 0.112, 0.105 (2s, 2 × CH₃) 28^(a) 6.81 (m) 5.84 (s) 5.19 (m)3.81 (m) 8.17 (s, H-8), 0.92 (s, ^(t)Bu), 0.103, 0.089 (2s, 2 × CH₃)29^(a) 30^(a) 7.00 (m) 5.86 (s) 5.29 (m) 3.87 (m) 8.78 (s, H-8), 8.22(s, H-2) 31^(a) 6.81 (m) 5.73 (d, J = 1.6 Hz) 4.96 (d, J = 2.8 Hz) 3.85(m) 8.19 (s, H-8), 0.91 (s, ^(t)Bu), 0.09, 0.084 (2s, 2 × CH₃) 32^(a)6.78 (m) 5.75 (s) 4.95 (m) 3.81 (m) 8.14 (s, H-8), 5.11 (s, NH₂), 0.89(s, ^(t)Bu), 0.076 (s, CH₃) 33^(a) 6.76 (m) 5.80 (s) 5.13 (ps t, J =4.4, 3.78 (m) 7.84 (s, H-8), 0.91 (s, ^(t)Bu), 0.093, 0.08 (2s, 2 × CH₃)4.8 Hz) 34^(a) 6.73 (ps t, J = 4.4, 5.80 (s) 5.09 (m) 3.78 (m) 7.84 (s,H-8), 5.12 (s, NH₂). 0.91 (s, ^(t)Bu), 0.096, 0.082 (s, CH₃) 4.8 Hz)35^(b) 6.90 (s) 6.08 (s) 4.91 (s) 3.63 (s) 8.40 (s, H-8), 8.17 (s, H-2),7.40 (s, NH₂), 5.22 (t, J = 5.6 Hz, OH) 36^(b) 6.89 (t, J = 4 Hz) 6.06(s) 5.14 (ps t, J = 3.6, 3.57 (m) 8.31 (s, H-8), 8.17 (s, H-2), 7.36 (s,NH₂), 4.97 (t, J = 6 Hz, OH) 4 Hz) 37^(b) 6.94 (m) 6.15 (t, J = 1.6 Hz)4.98 (s) 3.67 (s) 12.57 (br s, NH), 8.43 (s, H-8), 8.17 (s, H-2), 5.17(s, OH) 38^(b) 6.87 (ps t, J = 3.6, 6.06 (s) 5.13 (t, J = 3.6 Hz) 3.56(m) 8.26 (s, H-8), 8.09 (s, H-2) 4.4 Hz) ^(a)CDCl₃, ^(b)DMSO-d⁶

TABLE 4 No. H-1′ H-3′ H-4′ H-5′ Others 39^(b) 6.80 (s) 6.09 (ps t, J =1.2, 4.90 (s) 3.62 (m) 8.38 (s, H-8), 7.99, 7.92 (2br s, NH₂), 5.09 (t,1.6 Hz) J = 5.6 Hz, OH) 40^(b) 6.82 (m) 6.07 (d, J = 1.2 Hz) 5.12 (m)3.56 (m) 8.30 (s, H-8), 7.96 (2s, NH₂) 41^(b) 6.76 (s) 6.09 (s) 4.91 (s)3.60 (s) 8.38 (s, H-8), 7.07 (s, NH₂), 5.10 (s, OH) 42^(b) 6.72 (t, J =4 Hz) 6.06 (d, J = 1.2 Hz) 5.16 (ps t, J = 3.6, 3.60 (m) 8.30 (s, H-8),7.04 (s, NH₂), 4.88 (t, J = 6 Hz, OH) 4 Hz) 43^(b) 6.60 (s) 6.03 (d, J =1.2 Hz) 4.86 (s) 3.59 (s) 10.74 (br s, NH), 8.96 (s, H-8), 6.57 (s,NH₂), 5.08 (t, J = 5.2 Hz, OH) 44^(b) 6.62 (m) 6.01 (d, J = 1.6 Hz) 5.08(m) 3.56 (m) 7.82 (s, H-8), 6.57 (s, NH₂), 4.95 (t, J = 5.6 Hz, OH)^(b)DMSO-d⁶

TABLE 5 no. mp ° C. (solv)^(δ) [α]_(D2) deg formula anal. 10 syrupC₁₅H₂₃FN₂O₄Si C, H, N 11 syrup C₁₆H₂₅FN₂O₄Si C, H, N 12 144-146 (A)−20.47 (c 0.36, CHCl₃) C₂₂H₂₈FN₃O₄Si C, H, N 13 139-141 (A) +157.68(c.0.31, CHCl₃) C₂₂H₂₈FN₃O₄Si C, H, N 14 syrup C, H, N 15 syrup C, H, N16 161-162 (C) −13.412 (c 0.20, MeOH) C₉H₉FN₂O₄0.3H₂O C, H, N 17 136-137(E) +138.55 (c 0.14, MeOH) C₉H₉FN₂O₄0.2H₂O C, H, N 18 149-151 (D) −30.44(c 0.20, MeOH) C₁₆H₁₅FN₂O₅ C, H, N 19 116-118 (E) +132.42 (c 025, MeOH)C₁₆H₁₃FN₂O₅ C, H, N 20 200-202-dec (C) −54.89 (c 0.39, CHCl₃)C₁₆H₁₄FN₃O₄ C, H, N 21 170-172 (C) +136.38 (c 0.45, CHCl₃)C₁₆H₁₄FN₃O₄0.3H₂O C, H, N 22 198-200 dec (B) −21.31 (c 025, MeOH)C₉H₁₀FN₃O₃4H₂O C, H, N 23 120-121 (E) +159.15 (c 021, MeOH) C₉H₁₀FN₃O₃C, H, N 24 syrup C, H, N 25 syrup C, H, N 26 syrup C₁₆H₂₂FCIN₅O₂Si C, H,N 27 foam +9.80 (c 0.20, CHCl₃) C₁₆H₂₁F₂CIN₄O₂Si C, H, N 28 syrup+139.67 (c 0.18, CHCl₃) C₁₆H₂₁F₂CIN₄O₂Si C, H, N 29 C, H, N 30 foam C,H, N 31 180-182 (A) +13.33 (c 054, CHCl₃) C₁₆H₂₃F₂N₅O₂Si0.2aceton C, H,N 32 129-130 (A) +90.22 (c 0.23, CHCl₃) C₁₆H₂₃FCIN₅O₂Si C, H, N 33184-186 (A) +116.53 (c 0.13, CHCl₃) C₁₆H₂₃F₂N₅O₂Si0.3aceton C, H, N 34128-130 (A) +89.87 (c 0.15, CHCl₃) C₁₆H₂₃FCIN₅O₂Si C, H, N 35 188-189(C) −54.91 (c 0.17, MeOH) C₁₀H₁₀FN₅O₂0.2H₂O C, H, N 36 169-171 (C)+160.62 (c 0.19, MeOH) C₁₀H₁₀FN₅O₂0.3MeOH C, H, N 37 128-130 (E) −50.21(c 0.20, MeOH) C₁₀H₉FN₄O₃0.2H₂O C, H, N 38 >200 dec (C) +169.60 (c 0.20,MeOH) C₁₀H₉FN₄O₃0.3H₂O C, H, N 39 185-188 dec (B) −56.15 (c 0.16, MeOH)C, H, N 40 180 dec (B) +178.22 (c 0.10, MeOH) C, H, N 41 155-156 dec (B)+10.64 (c 0.17, MeOH) C, H, N 42 150-152 (B) +142.49 (c 0.17, MeOH) C,H, N 43 >200 dec (B) +24.42 (c 0.10, DMF) C, H, N 44 >210 dec (B) +58.68(c 0.10, DMF) C, H, N ^(δ)Solvents; A; EtOAc-hexanes, B; CH₂Cl₂-MeOH, C;CHCl₃-MeOH, D; THF-cyclohexane, E; lyophilyzed

Previously, the synthesis of 2′,3′-unsaturated D-nucleosides has beenaccomplished via elimination reaction starting from readily availablenucleoside analog, which involved a lengthy modification for individualnucleosides. Several groups reported D-2′-fluoro-2′,3′-unsaturatedpyrimidine nucleosides by the elimination of suitable 2′-fluorinatednucleoside analogs (Martin, J. A., et al., J. Med. Chem. 1990, 33,2137-2145; Stezycki R. Z., et al., J. Med. Chem. 1990, 33, 2150-2157).This strategy for the synthesis of L-Fd4N, however, is accompanied byadditional difficulties in the preparation of L-nucleosides as thestarting material. There are few examples of the synthesis of2′,3′-unsaturated purine nucleosides by direct condensation due to thelability of the 2,3-unsaturated sugar moiety under the couplingconditions in the presence of Lewis acid, except one case of thepyrimidine analog using a thiophenyl intermediate (Abdel-Medied, A.W.-S., et al., Synthesis 1991, 313-317; Sujino, K., et al., TetrahedronLett. 1996, 37, 6133-6136). In contrast to the 2,3-unsaturated sugarmoiety, the 2-fluoro-2,3-unsaturated sugar, which bears enhancedstability of glycosyl bond during the condensation with a heterocycle,was expected to become more suitable for the direct coupling reaction.Thus, (R)-2-fluorobutenolide 506, as a precusor for the key intermediate508, was chosen, which was prepared from L-glyceraldehyde acetonide 501.

Starting from L-glyceraldehyde acetonide, a mixture of (E)-502/(Z)-2(9:1 by ¹H NMR) was obtained via the Horner-Emmons reaction in thepresence of triethylα-fluorophosphonoacetate and sodiumbis(trimethylsilyl) amide in THF (Thenappan, A., et al., J. Org. Chem.,1990, 55, 4639-4642; Morikawa, T., et al., Chem. Pharm. Bull. 1992, 40,3189-3193; Patrick, T. B., et al., J. Org. Chem. 1994, 59, 1210-1212).Due to the difficulties in separating (E)-502/(Z)-502 isomers, themixtures were used in the following cyclization reaction under acidiccondition to give the desired lactone 503 and uncyclized diol 504. Theresulting mixture was converted to the silyl lactone 506 was subjectedto reduction with DIBAL-H in CH₂Cl₂ at 78° C. to give the lactol 507.The lactol 507 was treated with acetic anhydride to yield keyintermediate 508, which was condensed with silylated 6-chloropurineunder Vorburggen conditions to afford anomeric mixtures 509. Treatmentof 509 with TBAF in THF gave free nucleosides 510 and 511, which wasreadily separated by silica gel column chromatography. Adenine analogs512 and 513 were obtained by the treatment of compound 510 and 511 withmercaptoethanol and NaOMe a steel bomb at 90° C., respectively.Treatment of compounds 510 and 511 with mercaptoethanol and NaOMeafforded the inosine analogs 514 and 515, respectively. Thestereochemical assignment of these compounds was based on the NOESYspectroscopy (cross peak between H-1′ and H-4′ in B-isomer 512).

TABLE 7 Median Effective (EC₅₀) and Inhibitory (IC⁵⁰) Concentration ofL-2′- Fluoro-d4Adenine and Hypoxanthine against HIV-1 in PBM EC₉₀Cytotoxicity (μM) PMC CEM Compound EC₅₀ (μM) (PBM Cells Vero Cells CellsNo. (PBM Cells) Cells) IC₅₀ (μM) IC₅₀ (μM) IC₅₀ (μM) 512 1.515.1 >100 >100 >100 513 47.6 332 >100 >100 >100514 >100 >100 >100 >100 >100 515 >100 >100 >100 >100 >100 316(β) >100 >100 >100 >100 >100 317 (α) >100 >100 >100 >100 >100 318(β) >100 >100 >100 >100 >100 319 (α) >100 >100 >100 >100 >100 322 (β)0.51 4.3 >100 >100 >100 323 (α) >100 >100 >100 >100 >100 335 (β) 1.515.1 >100 >100 >100 336 (α) 47.6 332 >100 >100 >100 337(β) >100 >100 >100 >100 >100 338 (α) >100 >100 >100 >100 AZT 0.0040.04 >100 29.0 14.3Experimental Section.

Melting points were determined on a Mel-temp II laboratory device andare uncorrected. Nuclear magnetic resonance spectra were recorded on aBruker 250 and AMX400 400 MHz spectrometers with tetramethylsilane asthe internal reference; chemical shifts (δ) are reported in parts permillion (ppm), and the signals are described as s (singlet), d(doublet), t (triplet), q (quartet), br s (broad singlet), dd (doubletof doublet), and m (multiplet). UV spectra were obtained on a Beckman DU650 spectrophotometer. Optical rotations were measured on a JascoDIP-370 Digital Polarimeter. Mass spectra were measured on a MicromassInc. Autospec High Resolution double focussing sector (EBE) MSspectrometers. Infrared spectra were recorded on a Nicolet 510 FT-IRspectrometer. Elemental analyses were performed by Atlantic Microlab,Inc., Norcross, Ga. All reactions were monitored using thin layerchromatography on Analtech, 200 mm silica gel GF plates. Dry1,2-dichloroethane, dichloromethane, and acetonitrile were obtained bydistillation from CaH₂ prior to use. Dry THF was obtained bydistillation from Na and benzophenone when the solution became purple.

L-(S)-Glyceraldehyde acetonide (302)

A solution of L-gluconic-γ-lactone (175 g, 0.98 mol) in DMF (1 L) wascooled to 0 ° C. and p-toluenesulfonic acid (1.1 g, 5.65 mmol) was addedportionwise with stirring. To the resulting solution, 2-methoxypropene(87.7 g. 0.92 mol) was added dropwise through a dropping funnel at 0 °C. The reaction mixture was warmed up to room temperature and furtherstirred for 24 h. After the completion of the reaction, sodium carbonate(124 g) was added and the resulting suspension was vigorously stirredfor 3 hours. It is then filtered over glass filter and the filtrate isevaporated under vacuum. To the yellow residue, toluene (170 mL) isadded whereupon crystallization occurred. The solid was filtered bysuction, washed with hexanes/ethanol (9:1; 1 L), and dried to giveyellowish solid 301 (99.1 g, 65%).

To a stirred suspension of 5,6-O-isopropylidene-L-gulono-1,4-lactone(70.0 g, 0.32 mol) in water (270 mL), sodium metaperiodate (123 g, 0.58mol) was added portionwise at 0° C. over 30 min maintaining pH 5.5(adjusted by addition of 2 N NaOH). The suspension was stirred at roomtemperature for 2 hours, then saturated with sodium chloride andfiltered. The pH of the filtrate was adjusted to 6.5-7.0 and extractedwith dichloromethane (5 times 200 mL) and ethyl acetate (5 times 300mL). The combined organic layer were dried with anhydrous magnesiumsulfate, filtered and concentrated under reduced pressure (<20 ° C.).And then the resulting residue was distilled to give 302 (23.2 g, 69%)as a colorless oil; b.p. 49-51 ° C./ 16 Torr. [α]_(D)25-66.4 (c 6.3,benzene).

(E)/(Z)-Ethyl-3-[(R)-2,2-dimethyl-1,3-dioxolan-4-yl]-2-fluoroacrylate(E-303 and Z-303)

A solution of triethyl 2-fluorophosphonoacetate (39.2 g, 162 mmol) inTHF (70 mL) was cooled to −78 ° C. and a solution of sodiumbis(trimethylsilyl)amide (1.0 M solution in THF, 162 mL, 162 mmol) wasadded dropwise. The mixture was kept for 30 min at −78 ° C., then asolution of 303 (19.14 g, 147 mmol) in THF (70 mL) was added. Afterbeing stirred for 1 h at −78° C., the reaction mixture was treated withaqueous NH₄Cl and extracted with ether. The ether phase was washed withsaturated NaCl, dried over MgSO₄, filtered and evaporated. The residuewas chromatographed on silica gel to give E-303 and Z-303 (9:1 by ¹HNMR) as a pale yellowish oil (34.6 g, 97.9%). ¹H NMR (CDCl₃) δ1.34, 1.36(2t, J=8 Hz, —CH₂CH₃), 1.40, 1.45 (2s, —CH₃), 3.69 (m, H_(a)-5), 4.28(m, H_(b)-5, —CH₂CH₃), 5.02 (m, H-4), 5.40 (m, H-4), 6.02 (dd, J=8, 20Hz, H-3), 6.18 (dd, J=8, 32 Hz, H-3).

(R)-(+)-4-[(tert-Butyldimethylsilyloxy)methyl]-2-fluoro-2-buten-4-olide(307)

A solution of E-303 and Z-303 (19.62 g, 89.89 mmol) in 110 mL ofanhydrous EtOH was treated with 30 mL of conc. HCl and stirred at roomtemperature for 2 hr. The solvent was removed in vacuo and the residuewas coevaporated with Toluene (3*300 mL) to give the lactone 304 anduncyclized ester 305. The resulting yellowish syrup was used for nextreaction without further purification. t-Butyldimethylsilyl chloride(27.1 g, 180 mmol) was added to a mixture of 304, 305 and imidazole(12.3 g, 180 mmol) in CH₂Cl₂ (250 mL) and the reaction mixture wasstirred for 4 h at room temperature. The resulting mixture was washedwith water, dried (MgSO₄), filtered and concentrated to dryness. Theresidue was isolated by silica gel column chromatography using 4%EtOAc-hexanes as an eluent to give 307 (28.0 g, 70.2% from compound 302)as a white crystalline solid. mp 48-50 ° C.; [α]²⁸ _(D)+105.3 (c 1.60,CHCl₃); ¹H NMR (CDCl₃) δ 0.07, 0.08 (2s, 2×CH₃), 0.88 (s, ^(t)Bu), 3.88(m, 2H, H-5), 5.01 (m, 1H, H-4), 6.73 (ps t, 1H, J=4 Hz); Anal. Calcdfor C₁₀H₁₉FO₃Si: C, 53.63; H, 7.77. Found: C, 53.70; H, 7.75.

1-Acetyl-4-[(tert-butyldimethylsiloxy)methyl]-2-fluoro-2-buten-4-olide(309)

Lactone 307 (20.58 g, 83.54 mmol) was dissolved in 200 mL of CH₂Cl₂under nitrogen atmosphere, then the mixture was cooled to −78 ° C. and1.0 M solution of DIBAL-H in CH₂Cl₂ (125 mL) was added. The resultingmixture was stirred for 2 hours at −78 ° C. The cold mixture was treatedwith dilute nitric acid, washed with water, and dried (Na₂SO₄).Evaporation of the solvent gave anomers of 308 as a pale yellow oil(16.6 g, crude yield 80%), which was used for the next step withoutfurther purification.

Ac₂O (25 mL, 0.27 mol) was added to a solution of 308 and pyridine (22mL, 0.27 mol) in CH₂Cl₂ (200 mL) at 0 ° C. and the resulting mixture wasstirred for 16 hours. The reaction mixture was washed with dilute HCl,saturated NaHCO₃ solution, and brine. The combined organic layer wasdried, filtered, and concentrated to dryness. The residue was columnchromatographed (6.5% EtOAc/hexanes) to give 309 (12.6 g, 65%) as acolorless oil.

General Procedure for Condensation of Acetate 309 with Pyrimidine Bases.

A mixture of uracil (420 mg, 3.75 mmol), hexamethyldisilazane (15 mL)and ammonium sulfate (20 mg) was refluxed for 3 hours under nitrogen.The clear solution obtained was concentrated to dryness in vacuo. TMSOTf(0.7 mL, 3.14 mmol) were added to the solution of sugar 309 (728 mg,2.50 mmol)) and the silylated base in dry DCE (20 mL) at 0 ° C. Thereaction mixture was stirred for 2 hours under nitrogen, poured into acooled sat. NaHCO₃ solution (30 mL) and stirred for 15 min. Theresulting mixture was washed, dried (Na₂SO₄), filtered, and concentratedin vacuo. The crude product was purified by column chromatography (3%MeOH/CHCl₃) to give 310 (0.960 g, 2.73 mmol, 73%) as an inseparableanomeric mixture, which was used in the next step without separation.

1-[5-O-(tert-Butyldimethylsilyl)-2,3-dideoxy-2-fluoro-L-gycero-pent-2enofuranosyl]uracil(310)

UV (CHCl₃) λ_(max) 257.5 nm.; Anal. (C₁₅H₂₃FN₂O₄Si) C, H, N.

1-[5-O-(tert-Butyldimethylsilyl)-2,3-dideoxy-2-fluoro-L-gycero-pent-2-enofuranosyl]thymine(311)

Silylated thymine (242 mg, 1.92 mmol), 307 (500 mg, 1.72 mmol), andTMSOTf (0.5 mL, 225 mmol) were reacted for 2 h to give a mixture of 311,which was purified by silica gel column chromatography (3% MeOH/CHCl₃)as an inseparable anomeric mixture (0.392 g, 1.10 mmol, 64%). UV (CHCl₃)λ_(max) 262.0 nm. Anal.(C₁₆H₂₅FN₂O₄Si) C, H, N.

N⁶-Benzoyl-1-[5-O-(tert-butyldimethylsilyl)-2,3-dideoxy-2-fluoro-(a,b)-L-glycero-pent-2-enofuranosyl]cytosine(312 and 313)

Silylated N⁶-benzoyl cytosine (790 mg, 3.67 mmol), 307 (470 mg, 1.62mmol), and TMSOTf (0.5 mL, 2.25 mmol) were reacted for 2 h to givemixtures of 312 and 313, which were purified by silica gel column (30%EtOAc/hexane) to afford β anomer 312 (0.34 g, 0.76 mmol, 47.1%) as awhite solid and α anomer 313 chromatography (0.23 g, 0.52 mmol, 31.8%)as a white solid. 312: UV (CHCl₃) λ_(max) 260.5 nm; Anal.(C₂₂H₂₈FN₃O₄Si) C, H, N.; 513: UV (CHCl₃) λ_(max) 260.5 nm.; Anal.(C₂₂H₂₈FN₃O₄Si) C, H, N.

5-Fluoro-1-[5-O-(tert-butyldimethylsilyl)-2,3-dideoxy-2-fluoro-(a,b-L-gycero-pent-2-enofuranosyl]cytosine(314 and 315)

Silylated 5-fluorocytosine (300 mg, 2.32 mmol), 309 (360 mg, 1.24 mmol),and TMSOTf (0.4 mL, 1.86 mmol) were reacted for 2 h to give a mixture of314 and 315, which was purified by silica gel column chromatography (3%MeOH/CH₂Cl₂) to afford β anomer 314 as a white solid (168 mg, 37.8%) andα anomer 315 (121 mg, 27.1%) as a white solid. 314: UV (MeOH) λ_(max)281.5 nm; 315: UV (MeOH) λ_(max) 281.5 nm.

1-(2,3-Dideoxy-2-fluoro-(α,β-L-gycero-pent-2-eno-furanosyl)uracil (3.16and 317)

Tetra-n-butylammonium fluoride (0.6 mL, 0.6 mmol) was added to a mixtureof 310 (177 mg, 0.52 mmol) in THF (15 mL) and the reaction mixture wasstirred at room temperature for 15 min. The solvent was removed and theresidue was purified by silica gel column chromatography (2% MeOH/CHCl₃)to give β anomer 316 (52.8 mg, 0.23 mmol, 44.5%) and α anomer 317 (35.1mg, 0.15 mmol, 29.6%).

316: UV (H₂O) λ_(max) 261.0 nm (pH 7); Anal. (C₉H₉FN₂O₄.0.3H₂O) C, H, N.317: UV (H₂O) λ_(max) 261.0 nm (pH 7); Anal. (C₉H₉FN₂O₄.0.2H₂O) C, H, N.

1-(2,3-Dideoxy-2-fluoro-(α,β-L-gycero-pent-2-eno-furanosyl)thymine (318and 3.1.9)

Tetra-n-butylammonium fluoride (0.8 mL, 0.8 mmol) was added to a mixtureof 311 (240 mg, 0.67 mmol) in THF (10 mL) at 0 ° C. and the reactionmixture was stirred at room temperature at rt for 15 min. The solventwas removed and the residue was purified by silica gel columnchromatography (40% THF/cyclohexane) to give β anomer 318 (66.5 mg, 0.28mmol, 41%) and α anomer 319 (52.8 mg, 0.23 mmol, 26%).

318: UV (H₂O) λ_(max) 265.5 nm (pH 7); Anal. (C₁₀H₁₁FN₂O₄.0.4H₂O) C, H,N. 319:UV (H₂O) λ_(max) 266.0 nm (pH 7); Anal. (C₉H₉FN₂O₄.0.3H₂O) C, H,N.

N⁶-Benzoyl-1-(2,3-dideoxy-2-fluoro-β-L-gycero-pent-2-enofuranosyl)cytosine(320)

Tetra-n-butylammonium fluoride (1M in THF) (1 mL, 1 mmol) was added to asolution of the β anomer 312 (280 mg, 0.63 mmol) in THF (10 mL) and thereaction was allowed to stir at room temperature for 1 h. The reactionmixture was concentrated under the reduced pressure and the residue waspurified by flash silica gel column using 2.5% MeOH/CHCl₃ to give 320(218 mg, 0.66 mmol, 75%) as a white solid.

UV (MeOH) λ_(max) 260.5 nm. Anal. (C₁₆H₁₄FN₃O₄) C, H, N.

N⁶-Benzoyl-1-(2,3-dideoxy-2-fluoro-α-L-gycero-pent-2-enofuranosyl)cytosine(321)

Tetra-n-butylammonium fluoride (1M in THF) (1 mL, 1 mmol) was added to asolution of the α anomer 313 (280 mg, 0.63 mmol) in THF (10 mL) and thereaction was allowed to stir at room temperature for 1 h. The reactionmixture was concentrated under the reduced pressure and the residue waspurified by silica gel column chromatography using 2.5% MeOH/CHCl₃ togive 321 (145.8 mg, 0.44 mmol, 69%) as a white solid.

UV (MeOH) λ_(max) 260.5 nm. Anal. (C₁₆H₁₄FN₃O₄.0.3H₂O) C, H, N.

1-(2,3-dideoxy-2-fluoro-β-L-gycero-pent-2-enofuranosyl)cytosine (322)

A solution of the β anomer (67.60 mg, 0.204 mmol) in MeOH (5 mL) wastreated with NH₃/MeOH (10 mL saturated solution) and the reactionmixture was allowed to stir at room temperature until the disappearanceof starting material was observed (10 h). The reaction mixture wasconcentrated under reduced pressure and the residue was purified bypreparative TLC using 12% MeOH/CH₂Cl₂ as an eluent. The materialobtained from the plate gave 322 (43 mg, 93.1%) as a solid on trituationwith hexanes and diethylether.

UV (H₂O) λ_(max) 266.5 nm (pH 7); Anal. (C₉H₁₀FN₃O₃.0.4H₂O) C, H, N.

1-(2,3-dideoxy-2-fluoro-α-L-gycero-pent-2-enofuranosyl)cytosine (323)

A solution of the α anomer (65.90 mg, 0.199 mmol) in MeOH (5 mL) wastreated with NH₃/MeOH (10 mL saturated solution) and the reactionmixture was allowed to stir at room temperature until the disappearanceof starting material was observed (16 h). The reaction mixture wasconcentrated under reduced pressure and the residue was purified bypreparative TLC using 12% MeOH/CH₂Cl₂ as an eluent. The materialobtained from the plate gave 322 (42.5 mg, 94.5%) as a solid ontrituation with hexanes and diethylether.

UV (H₂O) λ_(max) 276.0 nm (pH 2), 267.0 nm (pH 7); Anal. (C₉H₁₀FN₃O₃) C,H, N.

5-Fluoro-1-(2,3-dideoxy-2-fluoro-β-L-gycero-pent-2-enofuranosyl)cytosine(324).

Tetra-n-butylammonium fluoride (1M in THF) was added to a solution ofthe β anomer 314 in acetonitrile and the reaction was allowed to stir atroom temperature for 1 h. The reaction mixture was concentrated underthe reduced pressure and the residue was purified by flash silica gelcolumn using 12% MeOH/CHCl₃ to give 324.

5-Fluoro-1-(2,3-dideoxy-2-fluoro-α-L-gycero-pent-2-enofuranosyl)cytosine(325)

Tetra-n-butylammonium fluoride (1M in THF) was added to a solution ofthe β anomer 315 in acetonitrile and the reaction was allowed to stir atroom temperature for 1 h. The reaction mixture was concentrated underthe reduced pressure and the residue was purified by flash silica gelcolumn using 12% MeOH/CHCl₃ to give 325.

General Procedure for Condensation of Acetate 309 with Purine Bases.

A mixture of 6-chloropurine (1.20 g, 7.75 mmol), hexamethyldisilazane(25 mL) and ammonium sulfate (catalytic amount) was refluxed for 4 hunder nitrogen. The clear solution was obtained was concentrated invacuo and the residue was dissolved in dry DCE (10 mL) and reacted witha solution of 307 (1.50 g, 5.17 mmol) in DCE (40 mL) and trimethylsilyltriflate (1.5 mL, 7.75 mmol) at room temperature. After stirring themixture for 1 h at room temperature under nitrogen, the reactionsolution was then poured into an ice cold saturated NaHCO₃ solution (20mL) and stirred for 15 min. The organic layer was washed with water andbrine, and dried over MgSO₄. The solvents were removed under reducedpressure and the residue was separated by silica gel columnchromatography using 12.5% EtOAc/hexanes to give anomeric mixture 326(1.25 g, 62.9%) as a syrup.

6-Chloro-9-[5-O-(tert-butyldimethylsilyl)-2,3-dideoxy-2-fluoro-L-gycero-pent-2-enofuranosyl]purine(326)

326: UV (MeOH) λ_(max) 265.0 nm; Anal. (C₁₆H₂₂ClFN₄O₂Si) C, H, N.

6-Chloro-2-fluoro-9-[5-O-(tert-butyldimethylsilyl)-2,3-dideoxy-2-fluoro-(α,β)-L-gycero-pent-2-enofuranosyl]purine(327 and 328)

A mixture of silylated 2-fluoro-6-chloropurine [prepared from 1.170 g(6.78 mmol) of 2-fluoro-6-chloropurine and dry DCE (40 mL) was stirredfor 16 h at room temperature. After work-up similar to that of 326,purification by silica gel column chromatography (12% EtOAc/hexanes)gave β anomer 327 (685 mg, 1.70 mmol, 30.0%) as a white foam and αanomer 328 (502 mg, 1.25 mmol, 22.1%) as an yellowish syrup.

327: UV (MeOH) λ_(max) 268.5 nm. Anal. (C₁₆H₂₁F₂Cl N₄O₂Si) C, H, N.,328: UV (MeOH) λ_(max) 269.0 nm. Anal. (C₁₆H₂₁F₂Cl N₄O₂Si) C, H, N.

6-Chloro-9-(2,3-dideoxy-2-fluoro-(α,β)-L-gycero-pent-2-enofuranosyl)purine(329 and 330)

A solution of 326 (1.2 g, 3.12 mmol) in dry CH₃CN (20 mL) was treatedwith TBAF (1 M solution in THF) (3.2 mL, 3.2 mmol) and stirred for 1 h.After evaporation of solvent, the dryness was purified by columnchromatography (3% MeOH/CHCl₃) to obtain β anomer 329 (296 mg, 35%) as awhite solid and α anomer 330 (380 mg, 45%) as a foam.

329: UV (MeOH) λ_(max) 265.0 nm.; 330: UV (MeOH) λ_(max) 265.0 nm.(332).

6-Amino-2-fluoro-9-[5-O-(tert-butyldimethylsilyl)-2,3-dideoxy-2-fluoro-β-L-gycero-pent-2-enofuranosyl]purine(331) and6-Chloro-2-amino-9-[-5-O-(tert-butyldimethylsilyl)-2,3-dideoxy-2-fluoro-β-L-gycero-pent-2-enofuranosyl]purine(332)

Dry ammonia was bubbled into a stirred solution of 327 (420 mg, 1.04mmol) in dry DME (35 mL) at room temperature overnight. The salts wereremoved by filtration and the filtrate was evaporated under reducedpressure. The residue was purified by silica gel column chromatography(25% EtOAc/hexanes) to give two compounds, 331 (114 mg, 0.30 mmol) as awhite solid and 332 (164 mg, 0.41 mmol) as a white solid.

331:UV (MeOH) λ_(max) 268.5 nm. Anal. (C₁₆H₂₃F₂N₅O₂Si0.2Acetone) C, H,N, 332:UV (MeOH) λ_(max) 307.5 nm. Anal. (C₁₆H₂₃FN₅O₂ClSi) C, H, N, Cl.

6-Amino-2-fluoro-9-[5-O-(tert-butyldimethylsilyl)-2,3-dideoxy-2-fluoro-α-L-gycero-pent-2-enofuranosyl]purine(333) and6-Amino-2-fluoro-9-[5-O-(tert-butyldimethylsilyl)-2,3-dideoxy-2-fluoro-α-L-gycero-pent-2-enofuranosyl]purine(334)

Dry ammonia was bubbled into a stirred solution of 333 (420 mg, 1.04mmol) in dry DME (35 mL) at room temperature overnight. The salts wereremoved by filtration and the filtrate was evaporated under reducedpressure. The residue was purified by silica gel column chromatography(25% EtOAc/hexanes) to give two compounds, 332 (150 mg, 0.38 mmol) as awhite solid and 333 (69.3 mg, 0.18 mmol) as a white solid.

333: UV (MeOH) λ_(max) 269.0 nm. Anal. (C₁₆H₂₃F₂N₅O₂Si0.3Acetone) C, H,N, 334: UV (MeOH) λ_(max) 309.5 nm. Anal. (C₁₆H₂₃F ClN₅O₂Si) C, H, N.

9-(2,3-dideoxy-2-fluoro-β-L-gycero-pent-2-enofuranosyl)adenine (335)

A solution of 329 (100 mg, 0.369 mmol) and saturated NH₃/MeOH (50 mL)was heated at 90 ° C. in a steel bomb for 24 h. After cooling to roomtemperature, the solvent was removed under reduced pressure and theresidual syrup was purified by column chromatography using 6% MeOH/CHCl₃as an eluent to give 335 (70 mg, 75%) as a white solid. 335: UV (H₂O)λ_(max) 258 nm (ε18,800) (pH 2), 258.5 nm (ε18,800) (pH 7), 258.5 nm(ε19,100) (pH 11). Anal. (C₁₀H₁₀FN₅O₂.0.2H₂O) C, H, N.

9-(2,3-dideoxy-2-fluoro-α-L-gycero-pent-2-enofuranosyl)adenine (336)

A solution of 330 (99 mg, 0.366 mmol) and saturated NH₃/MeOH (50 mL) washeated at 90 ° C. in a steel bomb for 24 h. After cooling to roomtemperature, the solvent was removed under reduced pressure and theresidual syrup was purified by column chromatography using 6% MeOH/CHCl₃as an eluent to give 336 (72 mg, 78%) as a white solid.

336: UV (H₂O) λ_(max) 258 nm (ε21,100) (pH 2), 259 nm (ε21,500) (pH 7),259 nm (ε22,600) (pH 11), Anal. (C₁₀H₁₀FN₅O₂.0.3MeOH) C, H, N.

9-(2,3-dideoxy-2-fluoro-β-L-gycero-pent-2-enofuranosyl)hypoxanthine(337)

A mixture of 329 (100 mg, 0.369 mmol), NaOMe (0.5 M solution in MeOH)(2.94 mL, 1.46 mmol) and HSCH₂CH₂OH (0.1 mL, 1.46 mmol) in MeOH (20 mL)was refluxed for 4 h under nitrogen. The reaction mixture was cooled,neutralized with glacial AcOH and evaporated to dryness under vacuum.The residue was purified by silica gel column chromatography (10%MeOH/CHCl₃) to afford 337 (74 mg, 80%) as a white solid. 37: UV (H₂O)λ_(max) 247 nm (ε12,400) (pH 2), 247.5 nm (ε13,000) (pH 7), 253 nm(ε13,100) (pH 11). Anal. (C₁₀H₉FN₄O₃.0.2H₂O) C, H, N.

9-(2,3-dideoxy-2-fluoro-α-L-gycero-pent-2-enofuranosyl)hypoxanthine(338)

A mixture of 330 (100 mg, 0.369), NaOMe (0.5 M solution in MeOH) (2.94mL, 1.46 mmol) and HSCH₂CH₂OH (0.1 mL, 1.46 mmol) in MeOH (20 mL) wasrefluxed for 4 h under nitrogen. The reaction mixture was cooled,neutralized with glacial AcOH and evaporated to dryness under vacuum.The residue was purified by silica gel column chromatography (10%MeOH/CHCl₃) to afford 338 (70 mg, 80%) as a white solid. 338: UV (H₂O)λ_(max) 247.5 nm (ε12,700) (pH 2), 247.5 nm (ε13,700) (pH 7), 252.5 nm(ε13,100) (pH 11). Anal. (C₁₀H₉FN₄O₃.0.3H₂O) C, H, N.

2-Fluoro-6-amino-9-(2,3-dideoxy-2-fluoro-β-L-gycero-pent-2-enofuranosyl)purine(339)

A solution of 31 (101 mg, 0.26 mmol) in dry acetonitrile (15 mL) wastreated with TBAF (1 M solution in THF) (0.35 mL, 0.35 mmol) and stirredfor 30 min. After evaporation of solvent, the dryness was purified bycolumn chromatography (9% CH₂Cl₂/MeOH) to obtain 339 (64.7 mg, 0.24mmol, 92.3%) as a white crystalline solid. UV (H₂O) λ_(max) 269.0 nm (pH7).

2-Fluoro-6-amino-9-(2,3-dideoxy-2-fluoro-α-L-gycero-pent-2-enofuranosyl)purine(340)

A solution of 333 (73.4 mg, 0.19 mmol) in dry acetonitrile (10 mL) wastreated with TBAF (1 M solution in THF) (0-25 mL, 0.25 mmol) and stirredfor 30 min. After evaporation of solvent, the dryness was purified bycolumn chromatography (9% CH₂Cl₂/MeOH) to obtain 340 (46.2 mg, 0.17mmol, 90.3%) as a white crystalline solid. UV (H₂O) λ_(max) 269.0 nm (pH7).

2-Amino-6-chloro-9-(2,3-dideoxy-2-fluoro-β-L-gycero-pent-2-enofuranosyl)purine(341)

A solution of 332 (143.5 mg, 0.40 mmol) in dry acetonitrile (15 mL) wastreated with TBAF (1M solution in THF) (0.6 mL, 0.60 mmol) and stirredfor 30 min. After evaporation of solvent, the dryness was purified bycolumn chromatography (5% CH₂Cl₂/MeOH) to obtain 341 (109 mg, 0.382mmol, 95.5%) as a white crystalline solid. UV (H₂O) λ_(max) 308.5 nm (pH7).

2-Amino-6-chloro-9-(2,3-dideoxy-2-fluoro-α-L-gycero-pent-2-enofuranosyl)purine(342)

A solution of 334 (145 mg, 0.36 mmol) in dry acetonitrile (10 mL) wastreated with TBAF (1 M solution in THF) (0.5 mL, 0.50 mmol) and stirredfor 30 min. After evaporation of solvent, the dryness was purified bycolumn chromatography (9% CH₂Cl₂/MeOH) to obtain 342 (99.9 mg, 0.35mmol, 96.4%) as a white crystalline solid. UV (H₂O) λ_(max) 309.0 nm (pH7).

9-(2,3-dideoxy-2-fluoro-β-L-gycero-pent-2-enofuranosyl)guanine (343)

A mixture of 341 (63.6 mg, 0.223 mmol), 2-mercaptoethanol (0.06 mL, 0.89mmol) and 1 N NaOMe (0.89 mL, 0.89 mmol) in MeOH (10 mL) was refluxedfor 5 h under nitrogen. The mixture was cooled, neutralized with glacialAcOH and concentrated to dryness under reduced pressure. The residue waspurified by column chromatography (12% CH₂Cl₂/MeOH) to obtain 343 (30.1mg, 0.113 mmol, 50.7%) as a white solid. UV (H₂O) λ_(max) 253.5 nm (pH7).

9-(2,3-dideoxy-2-fluoro-α-L-gycero-pent-2-enofuranosyl)guanine (344)

A mixture of 342 (59.3 mg, 0.208 mmol), 2-mercaptoethanol (0.07 mL, 1.04mmol) and 1 N NaOMe (1.04 mL, 1.04 mmol) in MeOH (10 mL) was refluxedfor 5 h under nitrogen. The mixture was cooled, neutralized with glacialAcOH and concentrated to dryness under vacuum. The residue was purifiedby column chromatography (12.5% CH₂Cl₂/MeOH) to obtain 344 (28.0 mg,0.105 mmol, 50.5%) as a white solid. UV (H₂O) λ_(max) 253.0 nm (pH 7).

Synthesis of cis-(±)-Carbocyclic d4 Cytosine Nucleosides and Their5′-Triphosphates

Referring to Scheme 11, starting from diethyl diallylmalonate (781), the4-carbethoxy-1,6-heptadiene (702) was synthesized in 78% yield (W. A.Nugent, J. Am. Chem. Soc, 1995, 117, 8992-8998). Compound 703 wassynthesized from, compound 702 in 71% yield (L. E. Martinez, J. Org.Chem., 1996, 61, 7963-7966), and compound 705 was synthesized fromcompound 704 in 43% yield (D. M. Hodgson, J. Chem. Soc. Perkin Trans. I,1994, 3373-3378). The key Intermediatecis-(±)-3-acetoxy-5-(acetoxymethyl)cyclopentene (708) can bealternatively synthesized from cyclopentadiene and formaldehyde inacetic acid using a Prins reaction (E. A. Saville-Stones, J. Chem. Soc.Perkin Trans. I, 1991, 2603-2604) albeit it suffers low yield andinseparable problems; or from a bicyclic lactone which was synthesizedby multiple steps through 4 steps (F. Burlina, Bioorg. Med. Chem. Lett.,1997, 7, 247-250). The latter methodology gave a chiral 708[(−)-enantiomer], although it needed to synthesized a chiral bicycliclactone. N⁴-Acetyl-5-fluorocytosine was synthesized from5-fluorocytosine and p-nitrophenyl acetate (A. S. Steinfeld, J. Chem.Research (M), 1979, 1437-1450).

Experimental Part

General. All reagents were used as received unless stated otherwise.Anhydrous solvents were purchased from Aldrich Chemical Co. Meltingpoints (M.p.) were determined on an Electrothermal digit melting pointapparatus and are uncorrected, ¹H and ¹³C NMR spectra were taken on aVarian Unity Plus 400 spectrometer at room temperature and reported inppm downfield from internal tetramethylsilane.

4-Carbethoxy-1,6-heptadiene (702)

A mixture of diethyl diallymalonate (701; 50 g, 208 mmol), sodiumcyanide (20.7 g, 422 mmol) and DMSO (166 mL) was heated at 160° C. for 6h. After being cooled to r.t., the mixture was added to 400 mL of waterand the product was extracted into hexane (4×100 mL). After evaporationof the solvent at reduced pressure, the residue was distilled (42-43°C./1 Torr) to give 27.34 g (78%) of 702 as a colorless liquid. ¹H NMR(400 MHz, CDCl₃) δ 5.80-5.70 (m, 2H, 2 CH═CH₂), 5.10-5.02 (m, 4B, 2CH═CH₂), 4.14 (q, 2H, J=7.2 Hz, OCH₂), 2.54-2.48 (m, 1H, CH), 2.41-2.34,2.29-2.23 (2m, 4H, 2 CH₂), 1.25 (t, J=7.2 Hz, 3H, CH₃).

(±)-3-Cyclopentenecarboxylic Acid, Ethyl Ester (703)

A flame-dried 500 mL flask was charged with 2,6-dibromophenol (1.20 g,4.76 mmol), tungsten oxychloride (0.813 g, 2.38 mmol), and anhydroustoluene (25 mL). The resulting suspension was heated at reflux undernitrogen for 1 h, and then the solvent was evaporated in vacuo. Thesolid residue was broken up with a spatula and dried in vacuo for 30min. To the residue were added toluene (160 mL), Et₄Pb (1.54 g, 4.76mL), and 702 (22 g, 131.0 mmol). The mixture was heated at 90° C. undernitrogen for 1.5 h. After being cooled to r.t., the mixture was filteredthrough a celite, and the celite was rinsed with t-BuOMe. The combinedfiltrates were washed with 1% NaOH soln, water, and brine, andconcentrated by evaporation at reduced pressure. The residue wasdistilled (37-38° C./1 Torr) to give 13.06 g (71%) of 703 as a colorlessliquid. ¹H NMR (400 MHz, CDCl₃) δ 5.67 (s, 2H, CH═CH), 4.14 (q, 2H, J=7.2 Hz, OCH₂), 3.11 (pentuplet, J=7.6 Hz, 1H, CH), 2.65 (d, J=7.6 Hz,4H, 2 CH₂), 1.27 (t J=7.2 Hz, 3H, CH₃).

(±)-1-(Hydroxymethyl)-3-cyclopentene (704)

To a cold (−78° C.) solution of 703 (7 g, 50 mmol) in dry THF (150 mL)was added LiAlH₄ (1 M soln in THF, 25 mL, 25 mmol), and the reactionsolution was stirred at −78° C. under argon for 4 h. Then the reactionsolution was allowed to warm to 0° C., and 2.5 mL of water, 2.5 mL of15% NaOH, and 7.5 mL of water were added sequentially. After warming tor.t, the precipitates were filtered through a celite, and the celite waswashed with hot EtOAc. The combined filtrates were washed with 0.1 KNaOH, and brine, dried (MgSO₄), filtered, concentrated and dried invacuo to give 4.294 g (84%) of 704 as a pale yellow liquid. ¹H NMR (400MHz, CDCl₃) δ 5.68 (s, 2H, 2 CH═CH), 3.57 (d, J=6.0 Hz, 2H, CH₂OH),2.54-2.48 (m, 3H, CH+CH₂), 2.15-2.10 (m, 2H, CH₂).

cis-(±)-4-(Hydroxymethyl)-1,2-epoxycyclopentane (705)

To a solution of 704 (930 mg, 9.1 mmol), and vanadyl acetylacetonate (10mg) in anhydrous CH₂Cl₂ (20 mL) was added t-BuO₂H [3 M soln in CH₂Cl₂,prepared from a mixture of t-BuO₂H (70% by weight in water, 41 mL, 0.3mol) and CH₂Cl₂ (59 mL) by drying (2×MgSO₄) and storage over 4Amolecular sieve, 10 mL, ˜30 mmol] dropwise. After stirring at r.t for 24h, aqueous Na₂SO₃ (15% soln, 60 mL) was added, and the mixture wasstirred at r.t. for 6 h. The organic layer was separated, washed withsat. NaHCO₃, and brine, and concentrated. The residue was purified byflash chromatography on silica gel eluting with hexane/EtOAc (2:1) togive 460 mg (43%) of 705 as a colorless liquid. ¹H NMR (400 MHz, CDCl₃)δ 3.54 (s, 2H, (CH)₂O), 3.49 (t, J=4.0 Hz, 2H, CH₂₀H), 2.95 (bs, 1H,OH), 2.44-2.40 (m, 1H, CH), 2.05-2.02 (m, 4H, 2 CH₂). ¹³C NMR (100 MHz,CDCl₃) δ 66.9 (d, (CH)₂O), 59.2 (t, CH₂OH), 36.5 (d, CH), 31.4 (t, 2CH₂).

cis-(±)-3-Acetoxy-5-(acetoxymethyl)cyclopentene (708)

To a solution of diphenyl diselenenide (2.70 g, 8.65 mmol) in anhydrousEtOH (100 mL) was added NaBH₄ in portions. The solution was stirreduntil the yellow color turned to colorless, and then a solution of 705(1.70 g, 14.4 mmol) in anhydrous THF (10 mL) was added. The reactionsolution was heated at reflux for 1 h under nitrogen, and then thesolvent was evaporated in vacuo. To the residue was added EtOAc (80 mL)and water (30 mL). The organic phase was separated, washed with brine,dried (MgSO₄), filtered, concentrated and dried in vacuo. The obtained(±)-1-hydroxy-4-hydroxymethyl)-2-(phenylselenenyl)-cyclopentane (706;light yellow oil) was used for the next reaction directly withoutfurther purification. To the crude product 706 were added anhydrousCH₂Cl₂ (60 mL), Et₃N (30 mL, 216 mmol), and DMAP (50 mg). The resultingsolution was cooled to 0° C., and Ac₂O (14.7 g, 144 mmol) was addeddropwise. After being stirred at r.t. under argon overnight, evaporationof the solvent provided(±)-1-acetoxy-4-(acetoxymethyl)-2-(phenylselenenyl)-cyclopentane (707;light yellow oil). To a cold (0° C.) solution of 707 in CH₂Cl₂ (50 mL)containing 3 drops of pyridine was added 30% H₂O₂ soln (20 mL) over aperiod of 5 min. After being stirred at 0° C. for 30 min and at r.t. foranother 30 min, the reaction mixture was diluted by addition of CH₂Cl₂(50 mL). The organic phase was separated, washed with water, sat.NaHCO₃, and brine, dried (MgSO₄), filtered, and concentrated byevaporation in vacuo. The residue was purified by flash chromatographyon silica gel eluting with 0-10% EtOAc in hexane to give 2.254 g (79%,for the three steps) of 708 as a pale brown liquid. ¹H NMR (400 MHz,CDCl₃) δ 6.01-6.00, 5.92-5.90 (2m, 2H, CH═CH), 5.66-5.64 (m, 1H, H-3),4.04 (d, J=6.8 Hz, 2H, CH₂O), 2.98-2.92 (m, 1H, H-5), 2.53-2.46 (m, 1H,H-4-a), 2.08, 2.04 (2s, 6H, 2 CH₃), 1.60-1.54 (m, 2H, H-4b). ¹³C NMR(100 MHz, CDCl₃) δ 171.1, 170.9 (2s, 2 C═O), 137.0, 131.4 (2d, CH═CH),79.2 (d, C-3), 67.4 (t, CH₂O), 43.7 (d, C-5), 33.4 (t, C-4), 21.3, 20.9(2q, 2 CH₃).

cis-(±)-Carbocyclic5′-O-acetyl-2′,3′-didehydro-2′,3′-dideoxy-5-fluorocytidine (709)

A suspension of 5-fluorocytosine (258 mg, 2 mmol) and NaH (58 mg, 2.4mmol) in anhydrous DMSO (15 mL) was heated in a pre-warmed oil bath at70° C. for 30 min. Then the resulting solution was cooled to r.t., andPd(PPh₃)₄ (73 mg, 0.063 mmol) and a solution of 708 (298 mg, 1.5 mmol)in anhydrous THF (2 mL) were added respectively. The reaction mixturewas stirred at 70° C. under argon for 3 days. After removal of thesolvent by evaporation in vacuo, the residue was treated with CH₂Cl₂ (50mL). The precipitates were filtered through a celite, and the celite waswashed with CH₂Cl₂. The combined filtrates were concentrated, and theresidue was purified by flash chromatography on silica gel eluting with0-5% MeOH in CH₂Cl₂ to give 40 mg (10%) of 709 as a light brown solid.Recrystallization from MeOH/CH₂Cl₂/hexane provided pure product as whitepowders. M.p. 182-184° C. ¹H NMR (400 MHz, CDCl₃) δ 7.43 (d, J=6.0 Hz,1H, H-6), 6.18-6.16 (m, 1H, H-3′), 5.83-5.81 (m, 1H, H-2′), 5.73-5.71(m, 1H, H-1′), 4.23-4.21, 4.08-4.04 (2m, 2H, CH₂O), 3.14-3.12 (m, 1H,H-4′), 2.92-2.84 (m, 1H, H-6′a), 2.08 (s, 3H, CH₃), 1.41-1.35 (m, 1H,H-6′b).

cis-(±)-CarbocyclicN⁴,5′-O-diacetyl-2′,3′-didehydro-2′,3′-dideoxy-5-fluorocytidine (710)

In an analogy manner to the procedure for 709, the title compound 710was prepared from 708 (560 mg, 2.828 mmol) andN⁴-acetyl-5-fluorocytosine (726 mg, 4.24 mmol): 560 mg (64%, brown oil).This crude product was used directly for the next reaction withoutfurther purification.

cis-(±)-CarbocyclicN⁴,5′-O-diacetyl-2′,3′-didehydro-2′,3′-dideoxycytidine (711)

In an analogy manner to the procedure for 709, the title compound 711was prepared from 708 (272 mg, 1.37 mmol) and N⁴-acetylcytosine (316 mg,2.06 mmol): 108 mg (27%) of white powders. M.p. 169.5-171.5° C. 3H NMR(400 MHz. CDCl₃) δ 8.80 (bs, 1H, NH), 7.72 (d, J= 6.8 Hz, 1H, H-6), 7.39(d, J=6.8 Hz, 1H, H-5), 6.19-6.17 (m, 1H, H-3′), 5.86-5.81 (m, 1H,H-2′), 5.77-5.75 (m, 1H, H-1′), 4.17-4.13, 4.07-4.02 (2m, 2H, CH₂O),3.18-3.10 (m, 1H, H-4′), 2.96-2.88 (m, 1H, H-6′a), 2.27, 2.06 (2s, 6H, 2CH₃), 1.43-1.37 (m, 1H, H-6′b). ¹³C NMR (100 MHz, CDCl₃) δ 170.8 (s, 2CO), 162.0 (s, C-4), 155.6 (s, C-2), 145.3 (d, C-6), 139.2 (d, C-3′),130.0 (d, C-25), 96.8 (d, C-5), 66.3 (t, C-5′), 62.8 (d, C-1′), 44.2 (d,C-4′), 34.7 (t, C-6), 25.0, 20.9 (2q, 2 CH₃).

cis-(±)-Carbocyclic 2′-3′-dehydro-2′,3′-dideoxy-5-fluorocytidine (712)

To a flask containing 709 (33 mg, 0.12 mmol) was added NaOMe (0.5 M solnin MeOH, 0.5 mL). The reaction solution was stirred at r.t. for 1 h, andthen the solvent was evaporated in vacuo. The residue was purified byflash chromatography on silica gel eluting with 5-10% MeOH in CH₂Cl₂ togive 17 mg (61%) of 712 as a light brown solid. Recrystallization fromMeOH/CH₂Cl₂/hexane provided pure product as white powders. M.p.205.5-206.0° C. ¹H NMR (400 MHz, DMSO-d₆) δ 7.66 (d, J=6.0 Hz, 1H, H-6),7.60, 7.40 (2bs, 2H, NH₂), 6.06-6.05 (m, 1H, H-3′), 5.68-5.65 (m, 1H,H-2′), 5.53-5.50 (m, 1H, H-1′), 4.77-4.75 (m, 1H, H-4′), 3.50-3.48,3.41-3.37 (2m, 2H, H-5′), 2.79-2.77 (m, 1H, H-6′a), 1.34-1.27 (m, 1H,H-6′b). ¹³C NMR (100 MHz, DMSO-d₆) δ 157.0 (d, J_(C-F)=11.9 Hz, C-4),154.0 (s, C-2), 139.2 (d, C-35), 135.8 (d, J_(C-F)=241.3 Hz, C-5), 130.2(d, C-2′), 126.8 (d, J_(C-F)=11.8 Hz, C-6), 63.5 (t C-5′), 61.3 (d,C-1′), 47.2 (d, C-4′), 33.3 (t, C-6′). MS (FAB) m/e 226 (MH⁺). Anal.(C₁₀H₁₂FN₃O₂) calcd C, 53:33; H, 5.37; N, 18.66. found C, 53.10; H,5.40; N, 18.44. In an analogy manner to the above procedure, the titlecompound 712 was also prepared from 710 (750 mg, 2.42 mmol): 320 mg(59%, white powders).

cis-(±)-Carbocyclic 2′,3′-didehydro-2′,3′-dideoxycytidine (713)

In an analogy manner to the procedure for 712, the title compound 713was prepared from 711 (75 mg, 0.257 mmol): 48 mg (90%, white solid).M.p. 200-201° C. ¹H NMR (400 MHz, DMSO-d₆) δ 7.40 (d, J=7.2 Hz, 1H,H-6), 7.03, 6.95 (2bs, 2H, NH₂), 6.07-6.05 (m, 1H, H-3′), 5.67 (d, J=7.2Hz, 1H, H-5), 5.65-5.64 (m, 1H, H-2′), 5.55-5.52 (m, 1H, H-1′),4.71-4.68 (m, 1H, H-4′), 3.43-3.36 (m, 2H, H-5′), 2.78-2.76 (m, 1H,H-6′a), 1.24-1.18 (m, 1H, H-6′b). ¹³C NMR (100 MHz, DMSO-d₆) δ 165.5 (s,C-4), 155.8 (s, C-2), 142.2 (d, C-6), 138.6 (d, C-3′), 130.5 (d, C-2′),93.7 (d, C-5), 63.9 (t, C-5′), 60.8 (d, C-1′), 47.3 (d, C-4′), 34.0 (t,C-6′). MS (FAB) m/e 208 (MH⁺). Anal. (C₁₀H₁₃N₃O₂) calcd D, 57.96; H,6.32; N, 20.28. found C, 57.35; H, 6.27; N, 20.02. HRMS (FAB) calcd for(C₁₀H₁₄N₃O₂) :208.1086; found 208.1088.

cis-(±)-Carbocyclic 2′,3′-didehydro-2′,3′-dideoxy-5-fluorocytidine5′-triphosphate, triethylhydrogenammonium salt (714)

To a solution of 712 (10 mg) in anhydrous DMF (0.3 mL) and pyridine (0.1ml) was added a 1 M solution of2-chloro-4H-1,3,2-benzodioxaphosphorin-4-one in anhydrous 1,4-dioxane(0.05 mL). The reaction solution was stirred at r.t. for 15 min. Then, asolution of 1 M pyrophosphoric acid-Bu₃N in anhydrous DMF (0.12 mL), andBu₃N (0.05 mL) was added sequentially. After stirring at r.t. foranother 15 min, a solution of I₂/H₂O/pyridine/THF was added to the abovesolution dropwise until the Iodine color persisted (about 0.5 mL), andthen the mixture was concentrated by evaporation in -vacuo. The residuewas dissolved in water (2 mL), washed with CH₂Cl₂ (3×1 mL), filtered,and purified by FPLC (column: HiLoad 26/10 Q Sepharose Fast Flow; bufferA: 0.01 M Et₃NHCO₃; buffer B: 0.7 M Et₃NHCO₃; flow rate: 10 mL/min;gradient: increasing buffer B from 0% at beginning to 10% at 4 min, thento 100% at 64 min). Collection and lyophilization of the appropriatefractions afforded 714 as a colorless syrup. HPLC [column: 100×4.6 mmRainin Hydropore SAX ionic exchange; buffer A: 10 mM NH₄H₂PO₄ hi 10%MeOH/H₂O (pH 5.5); buffer B: 125 mM NH₄H₂PO₄ in 10% MeOH/H₂O (pH 5.5);flow rate: 1.0 mL/min; gradient: increasing B from 0% at beginning to100% at 25 min] retention time: 11.9 min. MS (FAB) m/e 464 ([M-H]⁺).

cis-(±)-Carbocyclic 2′,3′-didehydro-2′,3′-dideoxycytidine 5′-phosphate(715)

In an analogy manner to the procedure for 714, the title compound 715was prepared from 713. HPLC (same conditions as above) retention time:12.1 min. MS (FAB) m/e 446 ([M-H]⁺).

Inhibitory Effect of (±)Carboxy-D4FC-triphosphate Against HIV-1 ReverseTranscriptase.

Extension assays were performed using a r(I)_(n)-(dC)₁₂₋₁₈ homopolymertemplate-primer (Pharmacia, Piscataway, N.J.) and the HIV-1 heterodimerp66/51 reverse transcriptase (RT, Biotechnology General, Rehovat,Israel). The standard reaction mixture (100 μl) contained 100 mM Trishydrochloride (pH 8.0), 50 mM KCl, 2 mM MgCl₂, 0.05 units/mlr(I)_(n)-(dC)₁₂₋₁₈, 5 mM DTT, 100 μg/ml Bovine Serum Albumin, and 1 μM3H-dCTP (23 Ci/mmol). 3TCTP (0.001-50 μM) was the positive control.Compounds were incubated 1 hr at 37° C. in the reaction mixture with 1unit HIV-1 RT. The reaction was stopped with the addition of an equalvolume of cold 10% TCA/0.05% sodium pyrophosphate and incubated 30minutes at 4° C. The precipitated nucleic acids were harvested ontofiberglass filler paper using a Packard manual harvester (Meriden,Conn.). The radiolabel uptake in counts per minute (cpm) was determinedusing a Packard 9600 Direct Beta counter.

IV. Anti-HIV Activity

In one embodiment, the disclosed compounds or their pharmaceuticallyacceptable derivatives or salts or pharmaceutically acceptableformulations containing these compounds are useful in the prevention andtreatment of HIV infections and other related conditions such asAIDS-related complex (ARC), persistent generalized lymphadenopathy(PGL), AIDS-related neurological conditions, anti-HIV antibody positiveand HIV-positive conditions, Kaposi's sarcoma, thrombocytopenia purpureaand opportunistic Infections. In addition, these compounds orformulations can foe-used prophylactically to prevent or retard theprogression of clinical illness in individuals who are anti-HIV antibodyor HIV-antigen positive or who have been exposed to HIV.

The ability of nucleosides to inhibit HIV can be measured by variousexperimental techniques. One technique, described in detail below,measures the inhibition of viral replication in phytohemagglutinin (PHA)stimulated human peripheral blood mononuclear (PBM) cells infected withHIV-1 (strain LAV). The amount of virus produced is determined bymeasuring the virus-coded reverse transcriptase enzyme. The amount ofenzyme produced is proportional to the amount of virus produced.

Antiviral and cytotoxicity assays: Anti-HIV-1 activity of the compoundsis determined in human peripheral blood mononuclear (PBM) cells asdescribed previously (Schinazi, R. F.; McMillan, A.; Cannon, D.; Mathis,R.; Lloyd, R. M. Jr.; Peck, A.; Sommadossi. J.-P.; St. Clair, M.;Wilson, J.; Furman, P. A.; Painter, G.; Choi, W.-B.; Liotta, D. C.Antimicrob. Agents Chemother. 1992, 36, 2423; Schinazi, R. F.;Sommadossi, J.-P.; Saalmann, V.; Cannon, D.; Xie, M. Y,; Hart, G.;Smith, G.; Hahn, E. Antimicrob. Agents Chemother. 1990, 34, 1061). Stocksolutions (20-40 mM) of the compounds were prepared in sterile DMSO andthen diluted to the desired concentration in complete medium.3′-azido-3′-deoxy thymidine (AZT) stock solutions are made in water.Cells are infected with the prototype HIV-1_(LA1) at a multiplicity ofinfection of 0.01. Virus obtained from the cell supernatant arequantitated on day 6 after infection by a reverse transcriptase assayusing poly(rA)_(n).oligo(dT)₁₂₋₁₈ as template-primer. The DMSO presentin the diluted solution (< 0.1%) should have no effect on the virusyield. The toxicity of the compounds can be assessed in human PBM, CEM,and Vero cells. The antiviral EC₅₀ and cytotoxicity IC₅₀ is obtainedfrom the concentration-response curve using the median effective methoddescribed by Chou and Talalay (Adv. Enzyme Regul. 1984, 22, 27).

Three-day-old phytohemagglutinin-stimulated PBM cells 10⁶ cells/ml) fromhepatitis B and HIV-1 seronegative healthy donors are Infected withHIV-1 (strain LAV) at a concentration of about 100 times the 50% tissueculture infectious dose (TICD 50) per ml and cultured in the presenceand absence of various concentrations of antiviral compounds.

Approximately one hour after infection, the medium, with the compound tobe tested (2 times the final concentration in medium) or withoutcompound, is added to the flasks (5 ml; final volume 10 ml). AZT is usedas a positive control.

The cells are exposed to the virus (about 2×10⁵ dpm/ml, as determined byreverse transcriptase assay) and then placed in a CO₂ incubator. HIV-1(strain LAV) is obtained from the Center for Disease Control, Atlanta,Ga. The methods used for culturing the PBM cells, harvesting the virusand determining the reverse transcriptase activity are those describedby McDougal et al. (J. Immun. Meth. 76, 171-183, 1985) and Spira et al.(J. Clin. Meth. 25, 97-99, 1987), except that fungizone was not includedin the medium (see Schinazi, et al., Antimicrob. Agents Chemother. 32,1784-1787 (1988); Id., 34:1061-1067 (1990)).

On day 6, the cells and supernatant are transferred to a 15 ml tube andcentrifuged at about 900 g for 10 minutes. Five ml of supernatant areremoved and the virus concentrated by centrifugation at 40,000 rpm for30 minutes (Beckman 70.1 Ti rotor). The solubilized virus pellet isprocessed for determination of the levels of reverse transcriptase.Results are expressed in dpm/ml of sampled supernatant. Virus fromsmaller volumes of supernatant (1 ml) can also be concentrated bycentrifugation prior to solubilization and determination of reversetranscriptase levels.

The median effective (EC₅₀) concentration is determined by the medianeffect-method (Antimicrob. Agents Chemother. 30, 491-498 (1986).Briefly, the percent inhibition of virus, as determined frommeasurements of reverse transcriptase, is plotted versus the micromolarconcentration of compound, lire EC₅₀ is the concentration of compound atwhich there is a 50% inhibition of viral growth.

Mitogen stimulated uninfected human PBM cells (3.8×10⁵ cells/ml) can becultured in the presence and absence of drug under similar conditions asthose used for the antiviral assay described above. The cells arecounted after 6 days using a hemacytometer and the trypan blue exclusionmethod, as described by Schinazi et al. Antimicrobial Agents andChemotherapy, 22(3), 499 (1982). The IC₅₀ is the concentration ofcompound which inhibits 50% of normal cell growth.

Table 7 provides data on the anti-HIV activity of selected compounds.Using this assay, it was determined that (±)-carbocyclic-D4FC-TP(2′,3′-unsaturated-5-fluorocytidine) exhibited an EC₅₀ of 0.40 μM, and(±)-carbocyclic-D4C-TP (2′,3′-unsaturated cytidine) exhibits an EC₅₀ of0.38 μM.

V. Anti-Hepatitis B Activity

The ability of the active compounds to inhibit the growth of hepatitisvirus in 2.2.15 cell cultures (HepG2 cells transformed with hepatitisvirion) can be evaluated as described in detail below.

A summary and description of the assay for antiviral effects in thisculture system and the analysis of HBV DNA has been described (Korba andMilman, 1991, Antiviral Res., 15:217). The antiviral evaluations areoptimally performed on two separate passages of cells. All wells, in allplates, are seeded at the same density and at the same time.

Due to the inherent variations in the levels of both intracellular andextracellular HBV DNA, only depressions greater than 3.5-fold (for HBVvirion DNA) or 3.0-fold (for HBV DNA replication intermediates) from theaverage levels for these HBV DNA forms in untreated cells are consideredto be statistically significant (P<0.05). The levels of integrated HBVDNA in each cellular DNA preparation (which remain constant on a percell basis in these experiments) are used to calculate the levels ofintracellular HBV DNA forms, thereby ensuring that equal amounts ofcellular DNA are compared between separate samples.

Typical values for extracellular HBV virion DNA in untreated cellsranged from 50 to 150 pg/ml culture medium (average of approximately 76pg/ml). Intracellular HBV DNA replication intermediates in untreatedcells ranged from 50 to 100 μg/pg cell DNA (average approximately 74pg/μg cell DNA). In general, depressions in the levels of intracellularHBV DNA due to treatment with antiviral compounds are less pronounced,and occur more slowly, than depressions in the levels of HBV virion DNA(Korba and Milman, 1991, Antiviral Res., 15:217).

The manner in which the hybridization analyses can be performed forthese experiments resulted in an equivalence of approximately 1.0 pg ofintracellular HBV DNA to 2-3 genomic copies per cell and 1.0 pg/ml ofextracellular HBV DNA to 3×10⁵ viral particles/ml.

Toxicity analyses were performed to assess whether any observedantiviral effects are due to a general effect on cell viability. Themethod used herein are the measurement of the uptake of neutral red dye,a standard and widely used assay for cell viability in a variety ofvirus-host systems, including HSV and HIV. Toxicity analyses areperformed in 96-well flat bottomed tissue culture plates. Cells for thetoxicity analyses are cultured and treated with test compounds with thesame schedule as described for the antiviral evaluations below. Eachcompound are tested at 4 concentrations, each in triplicate cultures(wells “A”, “B”, and “C”). Uptake of neutral red dye are used todetermine the relative level of toxicity. The absorbance of internalizeddye at 510 nm (A_(sin)) are used for the quantitative analysis. Valuesare presented as a percentage of the average A_(sin) values in 9separate cultures of untreated cells maintained on the same 96-wellplate as the test compounds.

VI. Anti-Hepatitis C Activity

Compounds can exhibit anti-hepatitis C activity by inhibiting HCVpolymerase, by inhibiting other enzymes needed in the replication cycle,or by other known methods. A number of assays have wen published toassess these activities.

WO 97/12033, filed on Sep. 27, 1996, by Emory University, listing C.Hagedorn and A. Reinoldus as inventors, and which claims priority toU.S. Ser. No. 60/004,383, filed on September 1995, describes an HCVpolymerase assay that can be used to evaluate the activity of thecompounds described herein. This application and invention isexclusively licensed to Triangle Pharmaceuticals, Inc., Durham, N.C.Another HCV polymerase assays has been reported by Bartholomeusz, etal., Hepatitis C virus (HCV) RNA polymerase assay using cloned HCVnon-structural proteins; Antiviral Therapy 1996:1(Supp 4) 18-24.

VI. Treatment of Abnormal Cellular Proliferation

In an alternative embodiment, the compounds are used to treat abnormalcellular proliferation. The compound can be evaluated for activity bytesting in a routine screen, such as that performed cost by the NationalCancer Institute, or by using any other known screen, for example asdescribed in WO 96/07413.

The extent of anticancer activity can be easily assessed by assaying thecompound according to the procedure below in a CEM cell or other tumorcell line assay. CEM cells are human lymphoma cells (a T-lymphoblastoidcell line that can be obtained from ATCC, Rockviile, Md.). The toxicityof a compound to CEM cells provides useful information regarding theactivity of the compound against tumors. The toxicity is measured asIC₅₀ micromolar). The IC₅₀ refers to that concentration of test compoundthat inhibits the growth of 50% of the tumor cells in the culture. Thelower the IC₅₉, the more active the compound is as an antitumor agent.In general, 2′-fluoro-nucleoside exhibits antitumor activity and can beused in the treatment of abnormal proliferation of cells if it exhibitsa toxicity in CEM or other immortalized tumor cell line of less than 50micromolar, more preferably, less than approximately 10 micromolar, andmost preferably, less than 1 micromolar. Drug solutions, includingcycloheximide as a positive control, are plated in triplicate in 50 μlgrowth medium at 2 times the final concentration and allowed toequilibrate at 37° C. in a 5% CO₂ incubator. Log phase cells are addedin 50 μl growth medium to a final concentration of 2.5×10³ (CEM andSK-MEL-28), 5×10³ (MMAN, MDA-MB-435s, SKMES-1, DU-145, LNCap), or 1×10⁴(PC-3, MCF-7) cells/well and incubated for 3 (DU-145, PC-3, MMAN), 4(MCF-7, SK-MEL-28, CEM), or 5 (SK-MBS-1, MDA-MB-435s, LNCaP) days at 37°C. under a 5% CO₂ air atmosphere. Control wells include media alone(blank) and cells plus media without drug. After growth period, 15 μl ofCell Titer 96 kit assay dye solution (Promega, Madison, Wis.) are addedto each well and the plates are incubated 8 hr at 37° C. in a 5% CO₂incubator. Promega Cell Titer 96 kit assay stop solution is added toeach well and incubated 4-8 hr in the incubator. Absorbance is read at570 nm, blanking on the medium-only wells using a Biotek Biokineticsplate reader (Biotek, Winooski, Vt.). Average percent inhibition ofgrowth compared to the untreated control is calculated, IC₅₀, IC₉₀,slope and r value are calculated by the method of Chou and Talalay. ChouT-C, Talalay P. Quantitative analysis of dose-effect relationships: Thecombined effects of multiple drugs or enzyme inhibitors. Adv EnzymeRegul 1984, 22:27-55.

The active compound can be administered specifically to treat abnormalcell proliferation, and in particular, cell hyperproliferation. Examplesof abnormal cell proliferation include, but are not limited to: benigntumors, including, but not limited to papilloma, adenoma, firoma,chondroma, osteoma, lipoma, hemangioma, lymphangioma, leiomyoma,rhabdomyoma, meningioma, neuroma, ganglioneuroma, nevus,pheochromocytoma, neurilemona, fibroadenoma, teratoma, hydatidiformmole, granuosa-theca, Brenner tumor, arrhenoblastoma, hilar cell tumor,sex cord mesenchyme, interstitial cell tumor, and thyoma as well asproliferation of smooth muscle cells in the course of development ofplaques in vascular tissue; malignant tumors (cancer), including but notlimited to carcinoma, including renal cell carcinoma, prostaticadenocarcinoma, bladder carcinoma, and adenocarcinoma, fibrosarcoma,chondrosarcoma, osteosarcoma, liposarcoma, hemangiosarcoma,lymphangiosarcoma, leiomyosarcoma, rhabdomyosarcoma, myelocyticleukemia, erythroleukemia, multiple myeloma, glioma, meningeal sarcoma,thyoma, cystosarcoma phyllodes, nephroblastoma, teratomachoriocarcinoma, cutaneous T-cell lymphoma (CTCL), cutaneous tumorsprimary to the skin (for example, basal cell carcinoma, squamous cellcarcinoma, melanoma, and Bowen's disease), breast and other tumorsinfiltrating the skin, Kaposi's sarcoma, and premalignant and malignantdiseases of mucosal tissues, including oral, bladder, and rectaldiseases; preneoplastic lesions, mycosis fungoides, psoriasis,dermatomyositis, rheumatoid arthritis, viruses (for example, warts,herpes simplex, and condyloma acuminata), molluscum contagiosum,premalignant and malignant diseases of the female genital tract (cervix,vagina, and vulva). The compounds can also be used to induce abortion.

In this embodiment, the active compound, or its pharmaceuticallyacceptable salt, is administered in an effective treatment amount todecrease the hyperproliferation of the target cells. The active compoundcan be modified to include a targeting moiety that concentrates thecompound at the active site. Targeting moieties can include an antibodyor antibody fragment that binds to a protein on the surface of thetarget cell, including but not limited to epidermal growth factorreceptor (EGFR), c-Esb-2 family of receptors and vascular endothelialgrowth factor (VEGF).

VII. Pharmaceutical Compositions

Humans suffering from any of the disorders described herein can betreated by administering to the patient an effective amount of theactive compound or a pharmaceutically acceptable derivative or saltthereof in the presence of a pharmaceutically acceptable carrier ordiluent. The active materials can be administered by any appropriateroute, for example, orally, parenterally, intravenously, intradermally,subcutaneously, or topically, in liquid or solid form. A prefer weightof the recipient per day. The effective dosage range of thepharmaceutically acceptable derivatives can be calculated based on theweight of the parent nucleoside to be delivered. If the derivativeexhibits activity in itself, the effective dosage can be estimated asabove using the weight of the derivative, or by other means known tothose skilled in the art.

The compound is conveniently administered in unit any suitable dosageform, including but not limited to one containing 7 to 3000 mg,preferably 70 to 1400 mg of active ingredient per unit dosage form. Aoral dosage of 50-1000 mg is usually convenient.

Ideally the active ingredient should be administered to achieve peak,plasma concentrations of the active compound of from about 0.2 to 70 pM,preferably about 1.0 to 10 μM. This may be achieved, for example, by theintravenous injection of a 0.1 to 5% solution of the active ingredient,optionally in saline, or administered as a bolus of the activeingredient.

The concentration of active compound in the drug composition will dependon absorption, inactivation, and excretion rates of the drug as well asother factors known to those of skill in the art. It is to be noted thatdosage values will also vary with fee severity of the condition to bealleviated. It is to be further understood that for any particularsubject, specific dosage regimens should be adjusted over time accordingto the individual need and the professional judgment of the personadministering or supervising the administration of the compositions, andthat the concentration ranges set forth herein are exemplary only andare not intended to limit the scope or practice of the claimedcomposition. The active ingredient may be administered at once, or maybe divided into a number of smaller doses to be administered at varyingintervals of time.

A preferred mode of administration of the active compound is oral. Oralcompositions will generally include an inert diluent or an ediblecarrier. They may be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Pharmaceutically compatible bindingagents, and/or adjuvant materials can be included as part of thecomposition.

The tablets, pills, capsules, troches and the like can contain any ofthe following ingredients, or compounds of a similar nature: a bindersuch as microcrystalline cellulose, gum tragacanth or gelatin; anexcipient such as starch or lactose, a disintegrating agent such asalginic acid, Primogel, or corn starch; a lubricant such as magnesiumstearate or Sterotes; a glidant such as colloidal silicon dioxide; asweetening agent such as sucrose or saccharin; or a flavoring agent suchas peppermint, methyl salicylate, or orange flavoring. When the dosageunit form is a capsule, it can contain, in addition to material of theabove type, a liquid carrier such as a fatty oil. In addition, dosageunit forms can contain various other materials which modify the physicalform of the dosage unit, for example, coatings of sugar, shellac, orother enteric agents.

The compound can be administered as a component of an elixir,suspension, syrup, wafer, chewing gum or the like. A syrup may contain,in addition to the active compounds, sucrose as a sweetening agent andcertain preservatives, dyes and colorings and flavors.

The compound or a pharmaceutically acceptable derivative or saltsthereof can also be mixed with other active materials that do not impairthe desired action, or with materials that supplement the desiredaction, such as antibiotics, antifungals, anti-inflammatories, or otherantivirals, including other nucleoside compounds. Solutions orsuspensions used for parenteral, intradermal, subcutaneous, or topicalapplication can include the following components: a sterile diluent suchas water for injection, saline solution, fixed oils, polyethyleneglycols, glycerine, propylene glycol or other synthetic solvents;antibacterial agents such as benzyl alcohol or methyl parabens;antioxidants such as ascorbic acid or sodium bisulfite; chelating agentssuch as ethylenediaminetetraacetic acid; buffers such as acetates,citrates or phosphates and agents for tire adjustment of tonicity suchas sodium chloride or dextrose. The parental preparation can be enclosedin ampoules, disposable syringes or multiple dose vials made of glass orplastic.

If administered intravenously, preferred carriers are physiologicalsaline or phosphate buffered saline (PBS).

In a preferred embodiment, the active compounds are prepared withcarriers feat will protect the compound against rapid elimination fromthe body, such as a controlled release formulation, including implantsand microencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation.

Liposomal suspensions (including liposomes targeted to infected cellswith monoclonal antibodies to viral antigens) are also preferred aspharmaceutically acceptable carriers. These may be prepared according tomethods known to those skilled in the art, for example, as described inU.S. Pat. No. 4,522,811 (which is incorporated herein by reference inits entirety). For example, liposome formulations may be prepared bydissolving appropriate lipid(s) (such as stearoyl phosphatidylethanolamine, stearoyl phosphatidyl choline, arachadoyl phosphatidylcholine, and cholesterol) in an inorganic solvent that is thenevaporated, leaving behind a thin film of dried lipid on the surface ofthe container. An aqueous solution of the active compound or itsmonophosphate, diphosphate, and/or triphosphate derivatives is thenintroduced into the container. The container is then swirled by hand tofree lipid material from the sides of the container and to disperselipid aggregates, thereby forming the liposomal suspension.

This invention has been described with reference to its preferredembodiments. Variations and modifications of the invention, will beobvious to those skilled in the art from the foregoing detaileddescription of the invention.

1. A process for the preparation of a 2′-fluoronucleoside comprising: a)obtaining a chiral non-carbohydrate sugar ring precursor; b) reactingthe non-carbohydrate sugar ring precursor with an electrophilic sourceof fluorine to form a 2-fluoro substituted lactone; c) reducing the2-fluoro substituted lactone to a 2-fluoro substituted lactol; d)converting the 2-fluoro substituted lactol to a 2-fluoro substitutedacetate; and e) reacting the 2-fluoro substituted acetate with a purineor pyrimidine base to provide a 2′-fluoronucleoside.
 2. The process ofclaim 1, wherein the chiral non-carbohydrate sugar ring precursor isderived from D- or L-glutamic acid.
 3. The process of claim 1, whereinthe chiral non-carbohydrate sugar ring precursor is a lactone.
 4. Theprocess of claim 3, wherein the lactone is (4S)-5-(protectedoxy)-pentan-4-olide.
 5. The process of claim 4, wherein the(4S)-5-(protected oxy)-pentan-4-olide is protected as atert-butyldiphenylsilyl ether.
 6. The process of claim 1, furthercomprising adding a base to the non-carbohydrate sugar ring precursor instep b) prior to reacting with the electrophilic source of fluorine. 7.The process of claim 6, wherein the base is lithiumbis(trimethylsilyl)amide (LiHMDS).
 8. The process of claim 1, whereinthe electrophilic source of fluorine isN-fluoro-(bis)benzenesulfonimide.
 9. The process of claim 1, wherein instep b) the non-carbohydrate sugar ring precursor is converted to anenolate before reacting with the electrophilic source of fluorine. 10.The process of claim 6, wherein the non-carbohydrate sugar ringprecursor and the electrophilic source of fluorine are in solutionbefore addition of the base.
 11. The process of claim 1, wherein thepurine or pyrimidine base in step e) is a pyrimidine base.
 12. Theprocess of claim 1, wherein the purine or pyrimidine base in step e) isa purine base.
 13. The process of claim 9, further comprising reactingthe enolate with an alkyl halide.
 14. The process of claim 1, whereinthe 2-fluoro substituted lactone is reduced with diisobutylaluminumhydride (DIBAL-H).
 15. The process of claim 1, wherein in step c) thelactol is reacted with acetic anhydride (AC₂O) to form the acetate. 16.The process of claim 15, the lactol is reacted in the presence of acatalyst.
 17. The process of claim 16, wherein the catalyst isdimethylaminopyridine (DMAP).
 18. The process of claim 1, wherein instep e) the acetate is reacted with a purine or pyrimidine base in thepresence of a Lewis acid catalyst.
 19. The process of claim 18, whereinthe Lewis acid catalyst is trimethylsilyl triflate, tin chloride ortitanium chloride.
 20. The process of claim 1, wherein the purine orpyrimidine base is protected as a silylated purine or pyrimidine base.21. The process of claim 1, wherein the pyrimidine base is cytosine,uracil, thymine, 5-fluorocytosine or N⁴-acetyl cytosine.
 22. The processof claim 4, further comprising deprotecting the 2′-fluoronucleoside. 23.The process of claim 22, wherein the 2′-fluoronucleoside is deprotectedwith tetrabutylammonium fluoride (TBAF) or ammonium fluoride.
 24. Theprocess of claim 1, wherein the 2′-fluoronucleoside is in theβ-configuration.
 25. The process of claim 1, wherein the2′-fluoronucleoside is in the α-configuration.
 26. The process of claim1, wherein the 2′-fluoronucleoside is a β-D-2′-fluoronucleoside.
 27. Theprocess of claim 1, wherein the 2′-fluoronucleoside is aα-L-2′-fluoronucleoside.